A method for inducing a hypometabolic state in a patient with a disease, and an apparatus for the same purpose.

Abstract

The present invention provides a method for inducing a hibernation-like state and a device therefor. The present invention provides a method for reducing a theoretically set temperature for the body temperature of a subject and / or a feedback gain of heat production in the subject or a method for inducing a hibernation-like state in the subject, said method being a chemical and physical method that comprises applying an excitatory stimulation to a pyroglutamylated RF-amide peptide (QRFP) producing-neuron. The present invention also provides a device to be used for performing this method.

Description

[Technical Field]

[0001] The present invention provides a method for inducing a hypometabolic state in a person with a disease, and an apparatus for that purpose. [Background technology]

[0002] In subjects with acute, severe illnesses, inducing a hypometabolic state (e.g., a hibernation-like state) can delay the progression of the disease or extend the time until death from the disease. Separately, hypothermia therapy has been developed for various diseases. For example, the effectiveness of hypothermia therapy for neonatal hypoxic-ischemic encephalopathy has been disclosed. 41 Currently, hypothermia is the only therapy for which efficacy has been recognized. However, studies have shown that hypothermia after cardiac arrest does not result in any difference in prognosis compared to temperature management therapy (maintaining a temperature of 36°C). 42 This study led to a decline in the proactive use of hypothermia therapy in adults.

[0003] Examples of low metabolic states include hibernation-like states. Homeothermic animals, birds, and mammals maintain a constant body temperature (T B ) ambient temperature (T A To maintain a higher, narrower range, the body consumes most of its energy for heat production. However, some mammals actively slow down their metabolism and enter a state known as hibernation in order to survive the food shortages of winter. The animals return to a normal state without any apparent tissue damage. 1,2 While mice do not hibernate, they exhibit a short-term metabolically reduced state known as diurnal dormancy if they can benefit from a decrease in basal metabolism. In both hibernation and diurnal dormancy, the reduction in energy expenditure is achieved primarily through a decrease in metabolism, which is influenced by two main factors: the theoretical set temperature (TR) and the negative feedback gain (H) of heat production. In diurnal dormant mice, TR remains close to normal, but H decreases to nearly one-tenth of normal, resulting in a TB that is considerably lower than TR. 3In contrast, during hibernation, both TR and H decrease significantly, enabling the maintenance of a hypometabolic state that is more efficient than daily torpor and capable of responding to fluctuations in ambient temperature 4,5 Numerous experiments have established that this active metabolic depression is regulated by the central nervous system 6 However, its neural mechanisms remain completely unknown. Elucidating the mechanisms of daily torpor and / or hibernation is a necessary step to develop methods for artificially inducing an artificial hibernation-like hypometabolic state in non-hibernating animals including humans 1,7 and will also be beneficial for long-distance space exploration in the future. Here, it was found that excitatory manipulation of a novel, biochemically defined neuronal population in the hypothalamus results in a very prolonged hypometabolism / hypothermia state in mice. In this state, the metabolic rate drops to less than one third, but unlike under anesthesia, the mice still respond to changes in ambient temperature. Furthermore, the mice spontaneously recovered from this state without any apparent abnormalities. This discovery represents an important insight into the mechanisms of hibernation and the development of methods for inducing artificial hibernation-like states. Artificial induction of hibernation-like states in rodents such as mice and rats has been reported in recent years 43 . [Summary of the Invention]

[0004] The present invention provides a method for inducing a hypometabolic state (preferably a hibernation-like state) in a subject having a disease and an apparatus therefor.

[0005] The inventors have found that a hibernation-like state can be induced in living, non-hibernating animals by applying excitatory stimulation to neurons expressing the pyroglutaminated RF amide peptide (QRFP) gene in the brain region of the hypothalamus, including the anterior ventral periventricular nucleus (AVPe), medial preoptic area (MPA), and periventricular nucleus (Pe). The inventors have also found that in animals with disease, inducing a hibernation-like state that reduces overall vital activity slows the progression and worsening of the disease and improves the survival rate of the animals. The inventors have also found a method to distinguish between a low-metabolic state (preferably a hibernation-like state) and death based on oxygen consumption.

[0006] According to the present invention, for example, the following inventions are provided. (1) A device for monitoring a hypometabolic state in a subject with a disease that is in a controlled hypometabolic state (preferably a hibernation-like state), A measuring unit that measures the oxygen concentration of exhaled and inhaled air from the subject, A calculation unit that calculates the oxygen consumption of the subject from the difference in oxygen concentration between the subject's exhaled and inhaled breath, It is equipped with an oxygen monitoring unit that monitors the oxygen consumption of the target over time, The apparatus may further include a display unit that shows the results of oxygen monitoring, or a low-metabolic state monitoring unit that estimates the subject is in a low-metabolic state (preferably a hibernation-like state) when oxygen consumption is lower than before the low-metabolic state, and estimates the subject has died or may have died when oxygen consumption is zero. (1') A device for monitoring a hypometabolic state (preferably a hibernation-like state) in a subject with a disease and a controlled hypometabolic state (preferably a hibernation-like state), A measuring unit that measures the oxygen concentration of exhaled and inhaled air from the subject, A calculation unit that calculates the oxygen consumption of the subject from the difference in oxygen concentration between the subject's exhaled and inhaled breath, A unit for estimating the possibility of a hypometabolic state (preferably a hibernation-like state) estimates that the subject has entered a hypometabolic state (preferably a hibernation-like state) or may have entered a hypometabolic state (preferably a hibernation-like state) based at least on a decrease in oxygen consumption (and preferably a theoretically set temperature) compared to the subject's oxygen consumption (and preferably a theoretically set temperature) before entering a hypometabolic state (preferably a hibernation-like state), When oxygen consumption becomes zero, the system estimates that the subject has died or is likely to have died, and A device including a device. (1'') A device for monitoring a hibernation-like state in a subject with a disease that is in a hibernation-like state, A measuring unit that measures the oxygen concentration of exhaled and inhaled air from the subject, A calculation unit that calculates the oxygen consumption of the subject from the difference in oxygen concentration between the subject's exhaled and inhaled breath, A unit for estimating the possibility of a hibernation-like state estimates that the subject has entered a hibernation-like state, or is likely to have entered a hibernation-like state, based at least on a decrease in oxygen consumption (and preferably theoretically set temperature) compared to the subject's oxygen consumption (and preferably theoretically set temperature) before entering a hibernation-like state, When oxygen consumption becomes zero, the system estimates that the subject has died or is likely to have died, and A device including a device. (1''') A device for monitoring a hibernation-like state in a subject with a disease that is in a hibernation-like state, A measuring unit that measures the oxygen concentration of exhaled and inhaled air from the subject, A calculation unit that calculates the oxygen consumption of the subject from the difference in oxygen concentration between the subject's exhaled and inhaled breath, A unit for estimating the possibility of a hibernation-like state estimates that the subject has entered a hibernation-like state, or is likely to have entered a hibernation-like state, based at least on the fact that both the oxygen consumption and theoretical target temperature have decreased compared to the oxygen consumption and theoretical target temperature of the subject before entering a hibernation-like state, When oxygen consumption becomes zero, the system estimates that the subject has died or is likely to have died, and The apparatus includes {preferably further comprising an output device (preferably a display device, preferably a display or speaker) for outputting the estimation results from the estimation unit}. (2) The apparatus described in (1) above, wherein the controlled low metabolic state is a hibernation-like state. (3) The apparatus according to (1) or (2) above, further comprising a determination unit that records fluctuations in oxygen consumption over a certain period of time for a subject in a stable low metabolic state, sets a predetermined range based on the fluctuations in oxygen consumption over that certain period, and determines whether the oxygen consumption is within or outside the predetermined range. (4) The apparatus according to any one of (1) to (3) above, further comprising an output device, wherein if the oxygen consumption exceeds the upper limit of a specified range, the output device outputs to the output device that the subject may be about to awaken from a controlled hypometabolic state; if the oxygen consumption falls below the lower limit of a specified range, the output device (preferably a display device, preferably a display) outputs to the output device (preferably a display device, preferably a display or speaker) that the subject is in a controlled hypometabolic state; and when the oxygen consumption is within a specified range, the output device (preferably a display device, preferably a display or speaker) outputs to the output device (preferably a display device, preferably a display or speaker) that the subject is in a controlled hypometabolic state. (5) The apparatus according to any one of (1''') and (2) to (4) above, further comprising a deep body thermometer, a body temperature storage unit for storing the body temperature measured by the thermometer, a calculation unit for calculating a theoretical set temperature, and a calculation unit for calculating a heat generation feedback gain (H). (6) A device for stimulating pyroglutaminated RF amidopeptide (QRFP)-producing neurons in one or more regions selected from the group consisting of the anterior ventral periventricular nucleus (AVPe), medial preoptic area (MPA), and periventricular nucleus (Pe) in the brain of a living subject, wherein the subject preferably has a disease. The aforementioned device is A control unit that transmits a control signal to control the generation of voltage, A voltage generating unit that receives a control signal from the control unit and generates a voltage, A stimulation probe having an electrical stimulation electrode distal to the voltage generating unit, wherein the stimulation probe is long enough to access QRFP-producing neurons from the brain surface, and generates electrical stimulation at the distal electrical stimulation electrode by the voltage from the voltage generating unit, An outside temperature gauge, A deep body thermometer and The exhaled gas analysis unit measures the oxygen concentration in the exhaled gas, A recording unit that records the measured ambient temperature and at least one value selected from the group consisting of core body temperature and oxygen concentration, A calculation unit that calculates the oxygen consumption of the subject from the difference in oxygen concentration between the subject's exhaled and inhaled breath, A hibernation possibility determination unit determines that the subject has entered a hibernation-like state, or is likely to have entered a hibernation-like state, based at least on a decrease in both oxygen consumption and theoretical setting temperature compared to the subject's oxygen consumption and theoretical setting temperature before entering a hibernation-like state, A mortality determination unit that determines whether a subject has died or is likely to have died, at least based on the fact that oxygen consumption is zero. A device including a device. (7) A device for stimulating pyroglutaminated RF amide peptide (QRFP)-producing neurons in one or more regions selected from the group consisting of the anterior ventral periventricular nucleus (AVPe), medial preoptic area (MPA), and periventricular nucleus (Pe) in the brain of a living subject, wherein the subject preferably has a disease. The aforementioned device is A control unit that transmits a control signal to control the release of QRFP-producing neuron-stimulating compounds, The storage section for the compound, A compound dispensing unit that receives a control signal from the control unit and dispenses the compound from the compound storage unit, A guide comprising a compound release port and a pathway for the compound to the release port, for delivering the compound to QRFP-producing neurons, An outside temperature gauge, A deep body thermometer and The exhaled gas analysis unit measures the oxygen concentration in the exhaled gas, A recording unit that records the measured ambient temperature and at least one value selected from the group consisting of core body temperature and oxygen concentration, A calculation unit that calculates the oxygen consumption of the subject from the difference in oxygen concentration between the subject's exhaled and inhaled breath, A hibernation possibility determination unit determines that a subject has entered a hibernation-like state, or is likely to have entered a hibernation-like state, based at least on a decrease in both oxygen consumption and theoretical temperature compared to the oxygen consumption and theoretical temperature of a subject in a non-hibernation-like state, A mortality determination unit that estimates that the subject has died or may have died, at least based on the fact that oxygen consumption is zero. A device including a device. (8) The apparatus according to (6) or (7) above, further comprising a determination unit that determines whether the subject is in a hypothermic state based on the ambient temperature and core body temperature recorded in the recording unit. (9) The apparatus according to any one of (6) to (8) above, further comprising a determination unit that determines whether or not the subject is in a hypometabolic state based on the ambient temperature recorded in the recording unit, core body temperature, and oxygen concentration. (10) The apparatus according to any one of (6) to (9) above, wherein the hibernation possibility determination unit further includes a determination unit that determines whether or not the subject is in a hibernation-like state based on the outside temperature recorded in the recording unit, core body temperature, and oxygen concentration. (11) The apparatus according to any one of (8) to (10) above, wherein the control unit transmits a control signal to continuously or intermittently stimulate GRFP-producing neurons until it is determined that the subject is in any one state selected from the group consisting of a hypothermic state, a hypometabolic state, and a hibernation-like state. (12) A method for lowering the theoretical set temperature of body temperature in a mammal, comprising providing excitatory stimulation to pyroglutaminated RF amido peptide (QRFP) producing neurons, wherein the mammal is suffering from a disease. (13) The method according to (12) above, wherein the QRFP-producing neurons are neurons from one or more regions selected from the group consisting of the anterior ventral periventricular nucleus (AVPe), the medial preoptic area (MPA), and the periventricular nucleus (Pe). (14) The method according to (12) or (13) above, wherein the excitatory stimulus is a stimulus selected from the group consisting of chemical stimuli, magnetic stimuli, and electrical stimuli. (15) A method for screening for substances that excitate pyroglutaminated RF amidopeptide (QRFP)-producing neurons located in the regions of the anterior ventral periventricular nucleus (AVPe), medial preoptic area (MPA), and periventricular nucleus (Pe) in mammals, preferably mammals with disease, The test compound is brought into contact with the QRFP-producing neurons, Measuring the excitation of the QRFP-producing neurons, Select a test compound that provides excitatory stimulation to the QRFP-producing neurons, Methods that include... (15a) A method for screening for substances that specifically provide excitatory stimulation to pyroglutaminated RF amidopeptide (QRFP)-producing neurons located in the regions of the anterior ventral periventricular nucleus (AVPe), medial preoptic area (MPA), and periventricular nucleus (Pe) in mammals, preferably mammals with disease, To express receptors in cells that are specifically expressed in QRFP-producing neurons, The process involves bringing the test compound into contact with the cells, Measuring the excitation of the QRFP-producing neurons, Select a test compound that provides excitatory stimulation to the QRFP-producing neurons, Methods that include... (15b) A method for testing a substance or a composition containing such substance (e.g., pharmaceutical preparations and pharmaceutical compositions, etc.) that specifically excitates pyroglutaminated RF amidopeptide (QRFP)-producing neurons located in the regions of the anterior ventral periventricular nucleus (AVPe), medial preoptic area (MPA), and periventricular nucleus (Pe) in mammals, preferably mammals with disease, To provide pyroglutaminated RF amido peptide (QRFP) producing neurons, Contacting the test compound or the composition with the cells, Measuring the excitation of the QRFP-producing neurons, By comparing the excitation of QRFP-producing neurons before and after contact with the test compound, it is determined whether the test compound or the composition provides excitatory stimulation to the QRFP-producing neurons. Methods that include... (15c) A method for testing a substance or a composition containing such substance (e.g., pharmaceutical preparations and pharmaceutical compositions, etc.) that specifically provides excitatory stimulation to pyroglutaminated RF amidopeptide (QRFP)-producing neurons located in the regions of the anterior ventral periventricular nucleus (AVPe), medial preoptic area (MPA), and periventricular nucleus (Pe) in mammals, preferably mammals with disease, The test compound is administered to the anterior ventral periventricular nucleus (AVPe), medial preoptic area (MPA), and periventricular nucleus (Pe) regions of mammals. Measuring the excitation (e.g., electrical potential) of QRFP-producing neurons, By comparing the excitation of QRFP-producing neurons before and after contact with the test compound or the composition, it is determined whether the test compound or the composition provides excitatory stimulation to the QRFP-producing neurons. Methods that include... (15d) A method for testing a test compound or a composition containing such compound (e.g., pharmaceutical preparations and pharmaceutical compositions, etc.) that induces hibernation in mammals, preferably mammals with a disease, The test compound or composition is administered to the anterior ventricular nucleus (AVPe), medial preoptic area (MPA), and periventricular nucleus (Pe) regions of a mammal (e.g., a non-human mammal). To confirm that the aforementioned mammals hibernate, Methods that include... (15e) The method described in (15d) above, From the correlation between core body temperature (e.g., intestinal temperature) and oxygen consumption in the aforementioned mammals (e.g., non-human mammals), the core body temperature (theoretical setting temperature) and ΔVO2 / ΔT when oxygen consumption is assumed to be 0 were determined. B To estimate (the feedback gain of heat generation), A method for indicating that a mammal has entered hibernation, wherein both the theoretical set temperature and the negative feedback gain of heat generation have decreased compared to before administration of the test compound or a composition containing the compound (e.g., pharmaceutical formulations and pharmaceutical compositions). (15f) A method for testing whether a test compound or a composition containing the compound (e.g., a pharmaceutical preparation and a pharmaceutical composition, etc.) induces hibernation in a mammal such as a human, preferably a mammal with a disease, To provide estimates of the theoretical target temperature and the heat generation feedback gain in a human being when the test compound or composition is administered to the anterior ventral periventricular nucleus (AVPe), medial preoptic area (MPA), and periventricular nucleus (Pe), and estimates of the theoretical target temperature and the heat generation feedback gain in a human being before administration, This includes comparing the estimated theoretical setting temperature and the estimated heat generation feedback gain before administration of the test compound or composition with the estimated theoretical setting temperature and the estimated heat generation feedback gain after administration to confirm whether both estimates decrease. A method indicating that the mammal hibernated, based on the fact that the estimated theoretical set temperature and the estimated heat generation feedback gain decreased after administration compared to before administration. (15g) A method for determining (predicting, estimating, and computationally calculating) whether a test compound or a composition containing said compound (e.g., pharmaceutical preparations and pharmaceutical compositions, etc.) induces hibernation in mammals such as humans, preferably mammals with a disease, To provide estimates of the theoretical target temperature and the heat generation feedback gain in a human being when the test compound or composition is administered to the anterior ventral periventricular nucleus (AVPe), medial preoptic area (MPA), and periventricular nucleus (Pe), and estimates of the theoretical target temperature and the heat generation feedback gain in a human being before administration, This includes comparing the estimated theoretical setting temperature and the estimated heat generation feedback gain before administration of the test compound or composition with the estimated heat generation feedback gain before administration to confirm whether both the estimated theoretical setting temperature and the estimated heat generation feedback gain after administration decrease. A method indicating that the mammal hibernated, based on the fact that the estimated theoretical set temperature and the estimated heat generation feedback gain decreased after administration compared to before administration. (15h) A method for determining (predicting, estimating, and computationally calculating) whether a test compound or a composition containing said compound (e.g., pharmaceutical preparations and pharmaceutical compositions, etc.) induces hibernation in mammals such as humans, preferably mammals with a disease, In mammals such as humans to which the test compound or composition is administered to the anterior ventral periventricular nucleus (AVPe), medial preoptic area (MPA), and periventricular nucleus (Pe), oxygen consumption and core body temperature are recorded under at least two different ambient temperature conditions, both before and after administration. To estimate the correlation between oxygen consumption and core body temperature both before and after administration, This includes determining, based on the estimated correlation, whether the degree of decrease in oxygen consumption when core body temperature decreases decreases after administration compared to before administration, and whether the estimated core body temperature, assuming oxygen consumption is 0, decreases after administration compared to before administration. A method for indicating that a mammal hibernated, wherein the degree of decrease in oxygen consumption when core body temperature decreases is lower after administration compared to before administration, and the estimated core body temperature, assuming oxygen consumption is 0, is lower after administration compared to before administration. (16) A method for determining (by testing, prediction, estimation, or computational calculation) whether a test compound or a composition containing said compound (e.g., pharmaceutical preparations and pharmaceutical compositions, etc.) induces hibernation in mammals such as humans, preferably mammals with a disease, In mammals such as humans to which the test compound or composition has been administered to the anterior ventral periventricular nucleus (AVPe), medial preoptic area (MPA), and periventricular nucleus (Pe), the oxygen consumption and core body temperature recorded under at least two different ambient temperature conditions before and after administration, respectively, are provided (or recorded). To estimate the correlation between oxygen consumption and core body temperature both before and after administration, This includes determining, based on the estimated correlation, whether the degree of decrease in oxygen consumption when core body temperature decreases decreases after administration compared to before administration, and whether the estimated core body temperature, assuming oxygen consumption is 0, decreases after administration compared to before administration. A method for indicating that a mammal hibernated, wherein the degree of decrease in oxygen consumption when core body temperature decreases is lower after administration compared to before administration, and the estimated core body temperature, assuming oxygen consumption is 0, is lower after administration compared to before administration. (17) A device for monitoring the state of hibernation, A recording unit records oxygen consumption and core body temperature recorded under at least two different ambient temperature conditions before and after administration in a mammal such as a human, preferably a diseased mammal, to which a test compound or a composition containing said compound (e.g., a pharmaceutical preparation and a pharmaceutical composition, etc.) has been administered to the regions of the anterior ventral periventricular nucleus (AVPe), medial preoptic area (MPA), and periventricular nucleus (Pe), respectively. The system includes a calculation unit that estimates the correlation between oxygen consumption and core body temperature before and after administration, and determines, based on the estimated correlation, whether the degree of decrease in oxygen consumption when core body temperature decreases decreases after administration compared to before administration, and whether the estimated core body temperature, assuming oxygen consumption is 0, decreases after administration compared to before administration. A determination unit determines that the mammal has entered hibernation if the degree of decrease in oxygen consumption when core body temperature decreases is lower after administration compared to before administration, and the estimated core body temperature assuming oxygen consumption is 0 is lower after administration compared to before administration. A device equipped with. (18) A method for treating a mammal having a disease, A method comprising inducing a controlled hypometabolic state in the animal. (19) The method described in (18) above, A method by which a controlled hypometabolic state is induced in the mammal in question, thereby delaying the progression of disease in the mammal. (20) The method described in (18) above, A method by which a controlled hypometabolic state is induced in a mammal in such a manner that the mortality rate of the mammal is reduced. (21) The method according to any one of (18) to (20) above, wherein the controlled hypometabolic state is a hibernation-like state. (22) The method according to any of (18) to (21) above, wherein a controlled hypometabolic state is induced by stimulating QRFP neurons. (23) The method described in any of (18) to (22) above, wherein the disease is a serious acute disease. (24) The method according to any of (18) to (23) above, further comprising observing the progress of the animal. (25) The method according to (24) above, wherein observing the progress includes measuring the change in the oxygen consumption of the animal over time. (26) The method according to (24) or (25) above, wherein observing the progress includes observing the progression of the disease in the animal. (27) The method described in any of (24) to (26) above, wherein the animal's condition is observed during transport to a medical institution or in the intensive care unit of the medical institution. (28) A method for testing the effectiveness of a treatment (for example, a treatment that induces a hypometabolic state), Using a diseased mammal for the procedure, Observe the treated mammals and Compared to a control group that did not receive the treatment, it is determined whether the treatment reduces the rate of disease progression, reduces mortality, or extends survival time. Methods that include... (29) The apparatus described in any of (1) to (6), (7) to (11) and (17) above, or the method described in any of (12) to (16) and (18) to (28) above, wherein the disease is an acute disease. (30) The apparatus described in any of (1) to (6), (7) to (11) and (17) above, or the method described in any of (12) to (16) and (18) to (28) above, wherein the disease is a chronic disease. (31) The apparatus or method described in (29) above, wherein the disease is a serious disease. (32) The apparatus or method described in (30) above, wherein the disease is a serious disease. (33) A vehicle equipped with any of the devices described in (1) to (6), (7) to (11), and (17) above. (34) An ambulance vehicle equipped with any of the devices described in (1) to (6), (7) to (11), and (17) above. [Brief explanation of the drawing]

[0007] [Figure 1a]Figures 1a–h relate to the activation of Qrfp-iCre neurons that reduce hypothalamic body temperature and energy expenditure. Figure 1a shows the strategies for chemogenetic excitation of iCre-positive neurons in Qrfp-iCre mice. [Figure 1b] Chemical excitation of iCre-positive cells in Qrfp-iCre mice was found to induce hypothermia, as measured by infrared thermography. Heterozygous (Q-het) or homozygous (Q-homozygous) Qrfp-iCre mice carrying the heterozygous Rosa26dreaddm3(M3) and / or Rosa26dreaddm4(M4) alleles were used in the experiment. [Figure 1c] Distribution of Q neurons in Qrfp-iCre mice. Depicted by GFP expression after injection of AAV10-DIO-GFP into the medial basal hypothalamus. Scale bar (horizontal image), 500 μm; inset, 100 μm; coronal image, 200 μm. Pe: periventricular nucleus, AVPe: anterior ventricular nucleus, MPA: medial preoptic area, LPO: lateral preoptic area, AHA: anterior hypothalamus, VMH: ventromedial hypothalamus, LHA: lateral hypothalamus, SON: suprathalamic nucleus, DMH: dorsomedial hypothalamus, TMN: tuberomammillary nucleus, MM: medial mammillary nucleus, SCN: suprachiasmatic nucleus, VOLT: vascular organs of the plate of terminalis, TC: optic nucleus, ARC: optic vein, optic nucleus of the third ventricle. [Figure 1d] Representative thermometer results showing surface body temperature of Q-hM3D mice. CNO was injected intraperitoneally at 0 hours. Note that the tail temperature rises at 0.5 hours (arrow). [Figure 1e] Fos immunostaining of slice specimens from Q-hM3D mice 90 minutes after CNO IP. Scale bar, 100 μm. [Figure 1f] Procedure for metabolic analysis by chemogenetic activation of Q neurons in Q-hM3D mice. [Figure 1g]Time course of hypothermia / metabolic decline after activation of Cre-positive neurons by DREADD. Purple strain, Q-hM3D mice; yellow strain, Qrfp-iCre mice injected with AAV10-DIO-hM3Dq-mCherry into the lateral hypothalamus; black strain, Qrfp-iCre mice injected with AAV-DIO-mCherry into the medial basal hypothalamus (negative control). [Figure 1h] Q neuron-induced hypometabolism (QIH) persists for several days and can be re-induced by CNO injection. The lines and shadows in b and g represent the mean and standard deviation for each group, respectively. [Figure 2a] Figures 2a-l show the results of histological and functional analysis of Q neuron projections. Figure 2a shows a strategy to visualize the axonal projection pattern of Q neurons by expressing GFP in Q neurons by injecting AAV-DIO-GFP into Qrfp-iCre mice. [Figure 2b] Distribution of GFP-positive Q neurons in AVPe, MPA, and Pe. scale bars, 100 μm. [Figure 2c] Distribution of axons originating from Q neurons. Scale bar: 100 μm. [Figure 2d] Using the ScaleS method, cropped images of brain images were revealed, showing Q neurons and DMH fibers in AVPe. [Figure 2e] In situ hybridization analysis showing that a population of Q neurons expresses Vgat and / or Vglut2 in Q-hM3D mice. Scale bar, 100 μm. [Figure 2f] High-magnification image of the rectangular region shown in Figure 2e. [Figure 2g] Single-color image of the rectangular region in Figure 2e. [Figure 2h] High-magnification images of rectangular regions 1-3 shown in Figure 2f, representing Q neurons expressing Vgat, Vglut2, or both. (1) Vgat+mCherry+; (2) Vglut2+mCherry+; (3) Vgat+Vglut2+mCherry+. [Figure 2i]The percentage of Vgat-positive neurons in mCherry-expressing cells (counted from 4 sections prepared from 2 mice) was 1291 out of 1997 cells, Vglut2 was 359 out of 1997 cells, and Vglut2 was 115 out of 1997 cells. Other mCherry-expressing cells did not express either Vgat or Vglut2. [Figure 2j] Strategies for photogenerative excitation of Q neurons or their axons in DMH and RPa, scale bar, 100 μm. [Figure 2k] Changes in body temperature measured by a thermographic camera during photogenerative excitation of Cre-positive cells or their axons in AVPe / MPA, or in DMH or RPa. Four shots of light stimulation are shown with blue arrowheads. Lines and shading indicate the mean and standard deviation for each group, respectively. The lower panel shows representative thermographic images obtained by excitation of Q neurons (AVPe / MPA). Note that the tail shows heat release 5 minutes after the first light stimulation (arrow). [Figure 2l] Estimated TS 30 minutes after the fourth photostimulation. Note that the effect of DMH fiber stimulation on Ts is almost equivalent to the effect of cell body excitation in AVPe / MPA. Pelvis, periventricular nuclei; AVPe, anterior ventricular Pe; VOLT, vascular organs of the lamina terminalis; MPA, medial preoptic area; VLPO, ventrolateral hypothalamus; PVN, paraventricular nucleus of the hypothalamus; SON, supraoptic nucleus; DMH, dorsomedial hypothalamus; TMN, tuberomammillary nucleus; MM, medial mammillary nucleus; LC, locus coeruleus; PAG, periaqueductal gray matter; LPB, lateral paranucleus; RVLM, paraventricular nucleus; 3rd globus pallidus; globus pallidus; ventricle. [Figure 3a] Q neuron-induced metabolic depression is accompanied by a decrease in the body temperature setpoint. Changes in TB and VO2 in QIH in various TAs. QIH was induced in Q-hM3D mice by CNO injection. Lines and shading indicate the mean and standard deviation for each group. [Figure 3b] Minimum TB (left) and VO2 (right) under normal and QIH conditions. [Figure 3c] A schematic diagram of the thermogenesis and heat loss pathways in mammals. Heat loss is proportional to the difference between TB in the TA and G factors. Thermogenesis is governed by the difference between TR and TA in the H factor. [Figure 3d] Relationship between TB-TA and VO2 in various TAs. The slope of the curve indicates G. The dots represent recorded data, the thick line is drawn from the median of the posterior G, and the thin line is a curve drawn from 500 G values ​​randomly selected from the posterior sample. [Figure 3e] The posterior distribution of the estimated G(e) and the difference in G(f) from QIH to the normal state. [Figure 3f] The posterior distribution of the estimated G(e) and the difference in G(f) from QIH to the normal state. [Figure 3g] The relationship between TB and VO2 at various TAs. The negative slope of the curve indicates H, and the x-intercept indicates TR. See Figure 3d for an explanation of the points and lines. [Figure 3h] The distribution of estimated H(h) and the difference in H from QIH to the normal state (i). [Figure 3i] The distribution of estimated H(h) and the difference in H from QIH to the normal state (i). [Figure 3j] Distribution of estimated TR(j) and the difference in TR from QIH to normal state(k). [Figure 3k] Distribution of estimated TR(j) and the difference in TR from QIH to normal state(k). [Figure 3l] Metabolic transitions of QIH within an individual. The top row shows the changes in animal posture at various TAs during QIH. The second column is the time expansion of the third column, and each shows the metabolic transition of a representative animal. Note that mice exhibit a curled-up posture during FIT at TA=28°C (B) and a stretched-out posture during QIH at TA=28°C (D). Even during QIH with the TA lowered to 12°C, animals adopted a curled-up posture, as in FIT (E), indicating that they were taking a position to avoid heat loss. [Figure 4a] Figures 4a–g demonstrate that Q neurons play a role in inducing fasting-induced dormancy in mice. Figure 4a shows strategies for suppressing Q neuron function. Left panel: experimental procedure. Right panel: TeTxLC-eYFP expression in AVPe / MPA as shown by immunostaining with anti-GFP antibody. Scale bar: 100 μm. [Figure 4b] Schematic diagram of the FIT experiment. [Figure 4c]Normal FIT was abolished by expressing TeTxLC in Q neurons. Note that these mice did not exhibit a rapid oscillatory decrease in metabolism. [Figure 4d] Minimum VO2 at 24–36 hours and 36–48 hours was compared between control mice and TeTxLC mice. Suppression of Q neurons blocked the VO2 decrease typically seen during FIT. The estimated difference in minimum VO2 between the control group and TeTxLC mice was [0.01, 0.80] ml / g / h at 24–36 hours and [0.36, 1.16] ml / g / h at 36–48 hours. The small standard deviations (SD) of TB and VO2 in TeTxLC mice indicate the involvement of Q neurons in abrupt metabolic changes, including oscillatory changes, during FIT. The ">" and "<" signs indicate whether the 89% HPDI or the standard deviation from TeTxLC to control mice of the difference in estimated minimums is negative or positive. [Figure 4e] FIT was induced in control, Qrfp-iCre heterozygous, and homozygous mice, demonstrating that deletion of the QRFP peptide does not affect FIT. [Figure 4f] A procedure for visualizing input neurons that make monosynaptic contact with Q neurons using a recombinant rabies virus vector. [Figure 4g] Distribution of input neurons in Q neurons. Arrows indicate originating cells. Scale bar, 100 μm. [Figure 4h] Brain region containing input neurons. Scale bar, 100 μm. Pe, periventricular nucleus; AVPe, anterior ventricular nucleus; MPA, medial preoptic area; VOLT, vascular organs of the lamina terminalis; MnPO, median preoptic area; VMPO, ventromedial preoptic area; VLPO, ventral hypothalamic area; PVN, paraventricular nucleus of the hypothalamus; TC, tuber; opt, optic tract; ac, anterior commissure; f, fornix; 3V, third ventricle. [Figure 5] Figure 5 shows a schematic diagram of the apparatus according to the first embodiment. [Figure 6] Figure 6 shows a schematic diagram of the apparatus according to the first embodiment. [Figure 7] Figure 7 shows a schematic diagram of the apparatus according to the second embodiment. [Figure 8]Figure 8 shows a schematic of additional configurations of the apparatus in the first and second embodiments. [Figure 9A] Figure 9B shows the scheme for an experiment in which sepsis model animals are observed for 48 hours with or without induction of hibernation (QIH). [Figure 9B] Figure 9B shows the time course of survival rates in sepsis model animals in which hibernation (QIH) was induced, and in sepsis model animals in which hibernation was not induced. [Figure 10] Figure 10 shows oxygen consumption graphs for a sepsis model, a sepsis model with hypometabolism-induced sepsis, and a typical QIH (hibernation) model. [Figure 11A] Figure 11A shows the experimental scheme for observing an animal model of acute renal failure for 48 hours, with or without inducing hibernation (QIH). [Figure 11B] Figure 11B shows the time course of survival rates in acute renal failure model animals that were induced to hibernate (QIH) or those that were not induced to hibernate. [Figure 12A] Figure 12A shows an example of the configuration of the apparatus (C) of the present invention. [Figure 12B] Figure 12B shows an example of the configuration of the apparatus (C) of the present invention. [Figure 12C] Figure 12C shows a flowchart for determining the possibility of a hibernation-like state and the state of death in the present invention. [Figure 12D] Figure 12D shows a flowchart for determining whether the oxygen consumption of a subject in a stable metabolic state is within or outside that range, based on the fluctuations in oxygen consumption within that range, according to the present invention. [Figure 12E] Figure 12E shows an example of the configuration of the apparatus (C) of the present invention. Detailed description of the invention

[0008] In this specification, “subjects” means humans and non-human mammals, such as rodents including mice, rats, guinea pigs, gerbils, and hamsters; laboratory animals including ferrets, rabbits, dogs, and miniature pigs; and non-human primates including monkeys, gorillas, chimpanzees, orangutans, and bonobos.

[0009] In some embodiments, the subject has a disease. In some embodiments, the subject has a chronic disease. In some embodiments, the subject has an acute disease. In some embodiments, the disease, chronic disease, or acute disease is a serious disease. In all embodiments of the present invention, the subject is preferably a subject having a disease. An acute disease is a disease in which symptoms appear suddenly and progress rapidly. Examples of acute diseases include sepsis, acute renal failure, acute pneumonia, myocardial infarction, cerebral infarction, cerebral hemorrhage, and hemorrhagic shock. A chronic disease is a disease other than an acute disease. Examples of chronic diseases include tumors, autoimmune diseases, and aging.

[0010] In this specification, the "hypothalamus" refers to the center located in the diencephalon that regulates endocrine and autonomic functions. In this specification, "Q neurons" refer to nerve cells located in the medial region of the hypothalamus, namely the anterior ventral periventricular nucleus (AVPe), medial preoptic area (MPA), and periventricular nucleus (Pe), which produce pyroglutaminated RF amide peptide (QRFP). Pyroglutaminated RF amide peptide (QRFP) is a neuropeptide identified as the endogenous ligand for the GPR103 receptor. QRFP is strongly expressed in the hypothalamus and is thought to be involved in the regulation of sleep and wakefulness, as it has been shown to have an effect of enhancing the arousal system.

[0011] In this specification, "T A " refers to the ambient temperature (°C) of the subject, and "T B " refers to core body temperature (°C), and "T R " represents the theoretical set temperature (°C). "VO2" represents the oxygen consumption of the subject. R is, T A When T is changed B We will find the correlation between and VO2, and T when VO2 is zero. B This is the body temperature required. B This refers to the internal body temperature, not the surface temperature which is affected by the outside temperature. For example, in humans, T BThis can be determined by the blood temperature in the rectum, esophagus, bladder, or pulmonary artery. The negative feedback gain (H) of heat generation indicates the heat generation efficiency, where H = ΔVO2 / ΔT B It is determined by [method].

[0012] In this specification, “hypometabolic state” refers to a controlled hypometabolic state, including a hibernation-like state. A controlled hypometabolic state can be induced by hypometabolic therapy. Hypometabolic therapy induces a state of reduced metabolism by decreasing the amount of oxygen required by tissues. Furthermore, the hypometabolic state is preferably controlled by the brain. A brain-controlled hypometabolic state can be induced by stimulating Q neurons in the brain. Furthermore, a brain-controlled hypometabolic state can be induced by the method of the present invention. Examples of hypometabolic therapy include inducing natural dormancy (e.g., diurnal dormancy, hibernation, Q neuron-induced hypometabolic dysregulation (QIH)) and similar states. A method for inducing a short-term hypometabolic state (dormancy) is to restrict food intake (e.g., inducing a state of starvation under conditions of an ambient temperature of 12-24°C (e.g., withholding food for 24 hours)). A short-term hypometabolic state can be preferably induced in rodents such as mice and Syrian hamsters. One method for inducing a longer-term low-metabolic state (hibernation) is to stimulate Q neurons. Stimulation of Q neurons may be effective in inducing a low-metabolic state in mammals that do not hibernate in nature (non-hibernating mammals). In low-metabolic therapy, hypothermia is a secondary consequence. In contrast, hypothermia therapy lowers body temperature by directly reducing it. Hypothermia therapy is a treatment method that directly lowers body temperature, for example, by rapid intravenous injection of a drug cooled to 4°C, and is therefore fundamentally different from low-metabolic therapy. In particular, mammals try to maintain a constant body temperature, so if the body is cooled as in hypothermia therapy, unless shivering is suppressed with muscle relaxants, extra metabolism is required, and metabolism tends to increase. Therefore, hypothermia therapy is fundamentally different from low-metabolic therapy. In this specification, "hibernation" refers to a hypothermic and low-metabolic state observed in mammals. "Daily torpor" refers to a short-term low-metabolic state. The difference between hibernation and daily torpor is that in daily torpor, T R While there is little decrease in H and a decrease in T R It differs in that both and H are significantly reduced. In this specification, "hibernation-like state" means T A As T decreases R This refers to a state in which both H and are significantly reduced. In this specification, "non-hibernating animals" refers to animals that do not have the ecology of hibernating during winter or when fasting.

[0013] In this specification, “exhaled breath” refers to the air exhaled by the subject. In this specification, oxygen concentration is an indicator of the amount of oxygen per unit volume. The unit of oxygen concentration may be, for example, % or mmHg. In this specification, “oxygen consumption” (VO2) is the amount of oxygen consumed per unit time calculated from the oxygen concentrations in exhaled and inhaled breath. Since oxygen consumption varies with body weight, it may be calculated after being corrected to a unit weight (e.g., per kg and per g). Oxygen consumption may be calculated per unit time (e.g., per minute or per hour).

[0014] The inventors have found that a hibernation-like state can be induced in living, non-hibernating animals by applying excitatory stimulation to pyroglutaminated RF amide peptide (QRFP)-producing neurons in one or more regions selected from the group consisting of the anterior ventral periventricular nucleus (AVPe), medial preoptic area (MPA), and periventricular nucleus (Pe) of the hypothalamus. The inventors have also found that in the case of diseased subjects, induction of a hypometabolic state (preferably a hibernation-like state) improves the survival rate of the subjects. By inducing a hypometabolic state (preferably a hibernation-like state), the subject's metabolism can be reduced, thereby delaying all life activities, including disease progression. Therefore, in the present invention, the subjects are those having a disease, particularly a disease that affects survival rate (e.g., a serious disease).

[0015] In this specification, "induction of a hypometabolic state in peripheral tissues" means inducing a hypometabolic state in peripheral tissues, thereby reducing the oxygen and nutrient demand in those peripheral tissues. The induction of a hypometabolic state in an individual according to the present invention may be accompanied by the induction of a hypometabolic state in peripheral tissues. For example, since QIH is accompanied by the induction of a hypometabolic state in peripheral tissues, in QIH, the individual's oxygen consumption decreases, respiration decreases, and heart rate also decreases. Although not bound by theory, this decrease in respiration and heart rate can be achieved by the brain controlling respiration and heart rate in accordance with the oxygen and nutrient demand of peripheral tissues. In contrast, anesthesia suppresses the entire brain and eliminates control itself, so anesthesia is completely different from the hypometabolic state in QIH in terms of the state of brain control over the body (and peripheral tissues). In contrast, hypothermia therapy forcibly lowers the body temperature of mammals. In hypothermia therapy, a hypometabolic state can occur in peripheral tissues due to forced hypothermia induction, but it cannot reduce metabolism as much as hypometabolic therapy. Furthermore, it is known that hypothermia therapy can actually cause shivering in muscle tissue, and in the absence of muscle relaxants, metabolism may even increase. In contrast, the hypometabolic state induced by hypometabolic therapy reduces oxygen consumption in peripheral tissues, which in turn causes the brain to reduce respiration and circulation, allowing metabolism to decrease beyond the limits achievable by hypothermia therapy. Thus, the hypometabolic state induced by hypometabolic therapy differs from the hypometabolic states induced by anesthesia or hypothermia therapy, which are not controlled by the brain, in that the hypometabolic and hypothermic state in peripheral tissues is caused by active control by the brain.

[0016] Accordingly, the present invention provides a method for inducing a hibernation-like state in a living, non-hibernating animal subject, comprising applying excitatory stimulation to pyroglutaminated RF amide peptide (QRFP)-producing neurons in one or more regions selected from the group consisting of the anterior ventral periventricular nucleus (AVPe), medial preoptic area (MPA), and periventricular nucleus (Pe) of the hypothalamus. In one preferred embodiment, the subject has a disease. In one preferred embodiment, the subject has a chronic disease. In one preferred embodiment, the subject has an acute disease. In one preferred embodiment, the disease, chronic disease, or acute disease is a serious disease. A serious disease is a disease that is life-threatening if treatment is delayed. An acute disease may be, for example, one or more selected from the group consisting of sepsis, acute renal failure, acute pneumonia, myocardial infarction, cerebral infarction, cerebral hemorrhage, and hemorrhagic shock. A chronic disease may be one or more selected from the group consisting of tumors, autoimmune diseases, and aging.

[0017] Excitatory stimuli can be induced by stimulating the brain using deep brain electrodes or by stimulating QRFP-producing neurons with activators.

[0018] According to the present invention, a device (hereinafter sometimes referred to as "the device of the present invention") is provided for stimulating pyroglutaminated RF amide peptide (QRFP)-producing neurons in one or more regions selected from the group consisting of the anterior ventral periventricular nucleus (AVPe), medial preoptic area (MPA), and periventricular nucleus (Pe) in the brain of a living subject. (A1) The apparatus of the present invention is A control unit that transmits a control signal to control the generation of voltage, A voltage generating unit that receives a control signal from the control unit and generates a voltage, A stimulation probe having an electrical stimulation electrode distal to the voltage generating unit, wherein the stimulation probe has sufficient length to access QRFP-producing neurons from the brain surface, and generates electrical stimulation at the distal electrical stimulation electrode by the voltage from the voltage generating unit. This may include the following. This allows the device of the present invention to electrically provide excitatory stimulation to QRFP-producing neurons. Alternatively, in terms of providing chemical stimulation instead of electrical stimulation, (A2) The apparatus of the present invention is A control unit that transmits a control signal to control the release of QRFP-producing neuron-stimulating compounds, The storage section for the compound, A compound release unit that receives a control signal from the control unit and releases the compound from the compound storage unit. It may include. (B) The apparatus of the present invention is An outside temperature gauge, A deep body thermometer and The exhaled gas analysis unit measures the oxygen concentration in the exhaled gas, A recording unit that records the measured ambient temperature and at least one value selected from the group consisting of core body temperature and oxygen concentration, It may further include the following: In the configuration of (B) of the apparatus of the present invention, the above target is the ambient temperature (T A Along with the decrease in ), core body temperature (T B It is possible to investigate whether the oxygen consumption of the target oxygen decreases, and to determine the target oxygen consumption from the exhaled gas analysis results, and to determine the theoretical set temperature (T R The negative feedback gain (H) of heat generation can be determined. This allows the apparatus of the present invention to determine whether or not the subject has been induced into a hibernation-like state.

[0019] The apparatus (C) of the present invention is as shown in Figure 12A, A calculation unit 205 calculates the oxygen consumption of the subject from the difference in oxygen concentration between the exhaled and inhaled air from the subject; A unit 206 for determining the possibility of a hypometabolic state (preferably a hibernation-like state) determines, at least based on a decrease in oxygen consumption compared to the oxygen consumption of the subject before entering a hypometabolic state (preferably a hibernation-like state), that the subject has entered or may have entered a hypometabolic state (preferably a hibernation-like state); and / or, A mortality determination unit 207 determines that the subject has died or is likely to have died, based at least on the subject's oxygen consumption, the carbon dioxide concentration in the subject's exhaled breath, the waveform of the subject's electrocardiogram, or the subject's blood pressure being zero; The apparatus may further include the carbon dioxide concentration in the subject's exhaled breath, the waveform of the subject's electrocardiogram, or the subject's blood pressure data, which can be input to the apparatus of the present invention by an emergency monitor that acquires the subject's electrocardiogram and blood pressure. The carbon dioxide concentration in the subject's exhaled breath can also be input to the apparatus of the present invention by an exhaled carbon dioxide partial pressure monitor. The apparatus (C) may further include a determination unit 240 that takes into account the determinations from a determination unit 206 for the possibility of a hypometabolic state (preferably a hibernation-like state) and a determination unit 207 for the possibility of death, respectively, and determines whether the subject is in a hibernation state or a death state. The determination unit 240 is connected to an output device 250 (e.g., a display or speaker), and the result can be output from the output device 250. Thus, the apparatus (C) may further include an exhaled breath measurement unit 201 for measuring the oxygen concentration in exhaled breath and an inspiratory breath measurement unit 202 for measuring the oxygen concentration in inhaled breath. The determination unit 240 may determine the state of the subject according to, for example, the flowchart shown in Figure 12C. The apparatus (C) of the present invention may record fluctuations in oxygen consumption over a certain period of time for an object in a stable low-metabolism state (preferably a hibernation-like state), and output a notification from the output device 250 when the fluctuation falls outside a specified range. Here, when the fluctuation is within the specified range, it can be evaluated that a stable low-metabolism state is being maintained, and when it falls outside the specified range, it can be evaluated that the object has emerged from a stable low-metabolism state. The specified range can be determined based on fluctuations in oxygen consumption over a certain period of time. For example, the average value of the fluctuations in oxygen consumption over a certain period of time can be obtained, and the range between the average value × lower specified value and the average value × upper specified value can be set as the specified range. The lower specified value can be, for example, 0.7 or less, 0.6 or less, 0.5 or less, 0.4 or less, 0.3 or less, 0.2 or less, or 0.1 or less. The upper default values ​​can also be, for example, 1.3 or higher, 1.4 or higher, 1.5 or higher, 1.6 or higher, 1.7 or higher, 1.8 or higher, 1.9 or higher, 2 or higher, 2.1 or higher, 2.2 or higher, 2.3 or higher, 2.4 or higher, 2.5 or higher, 2.6 or higher, 2.7 or higher, 2.8 or higher, 2.9 or higher, or 3 or higher.The lower and upper default values ​​can be 0.7 and 1.3, 0.6 and 1.4, 0.5 and 1.5, 0.4 and 1.6, 0.3 and 1.7, 0.2 and 1.8, and 0.1 and 1.9, respectively. For this reason, the device (C) may further include a determination unit 241 that records fluctuations in oxygen consumption over a certain period of time for an object in a stable low metabolic state (preferably a hibernation-like state), sets a predetermined range based on the fluctuations in oxygen consumption over that period (for example, based on the upper and lower limits of oxygen consumption over that period, or the average value), and determines whether the oxygen consumption is within or outside the predetermined range. For example, the upper limit of the predetermined range can be determined based on the upper limit of the fluctuations in oxygen consumption over the aforementioned period. For example, the upper limit of the specified range can be set by a first percentage higher than the upper limit of the fluctuation in oxygen consumption over a certain period, and the first percentage can be 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 100% or more. For example, the lower limit of the specified range can be determined based on the lower limit of the fluctuation in oxygen consumption over a certain period. For example, the lower limit of the specified range can be set by a second percentage lower than the lower limit of the fluctuation in oxygen consumption over a certain period, and the second percentage can be 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 100% or more. If the oxygen consumption exceeds the upper limit of the specified range, it means that the subject may be about to awaken from a controlled hypometabolic state, and if the oxygen consumption falls below the lower limit of the specified range, it means that the subject may be about to transition to an even lower metabolic state than the controlled hypometabolic state (for example, about to die). Therefore, it is preferable that the result of the determination unit 241's determination of whether the oxygen consumption is within or outside the specified range is output to the output device 250. The determination unit 241 may determine the subject's state according to, for example, the flowchart shown in Figure 12D. The determination result of the determination unit 240 and the determination result of the determination unit 241 may be displayed simultaneously on the output device 250.Here, if the fluctuation in oxygen consumption of a subject in a low metabolic state falls within ±20%, ±15%, ±10%, or ±5% of the above-mentioned range of fluctuation in oxygen consumption over a certain period, the subject can be evaluated as being in a stable low metabolic state.

[0020] In one embodiment, the above-mentioned device (C) is A program that calculates oxygen consumption by determining the difference in oxygen concentration between exhaled and inhaled air from the subject, A program for determining the possibility of a hypometabolic state, which determines that a subject has entered or is likely to enter a hypometabolic state (preferably a hibernation-like state) based at least on a decrease in oxygen consumption compared to the subject's oxygen consumption before entering a hypometabolic state (preferably a hibernation-like state), A program for determining the likelihood of death determines that a subject has died or is likely to have died, based at least on the subject's oxygen consumption, the carbon dioxide concentration in the subject's exhaled breath, the waveform of the subject's electrocardiogram, or the subject's blood pressure being zero. The apparatus (C) has an arithmetic circuit 260 (e.g., a processor) capable of executing the following. The apparatus (C) transmits the decision result from the arithmetic circuit to an output device 250, which can output the result. In a preferred embodiment, for example, as shown in Figure 12D, the arithmetic circuit 260 of the apparatus (C) may further execute a program that records fluctuations in oxygen consumption over a certain period of time of an object in a stable low-metabolism state (preferably a hibernation-like state), determines a specified range, and then determines whether the oxygen consumption is within or outside the specified range. The arithmetic circuit 260 of the apparatus (C) can output the decision result of the program that determines whether the oxygen consumption is within or outside the specified range from the output device 250. The output result may be displayed on a display, or output as sound (e.g., a warning sound) from a speaker, or displayed on a display and output as sound (e.g., a warning sound) from a speaker. These programs may be executed by one or more arithmetic circuits.

[0021] In one embodiment, the above-mentioned device (C) is One or more processors and memory and It comprises one or more programs, the one or more programs being stored in the memory and configured to be executable by the one or more processors, and the one or more programs are A program that calculates oxygen consumption by determining the difference in oxygen concentration between exhaled and inhaled air from the subject, A program for determining the possibility of a hypometabolic state, which determines that a subject has entered or is likely to enter a hypometabolic state (preferably a hibernation-like state) based at least on a decrease in oxygen consumption compared to the subject's oxygen consumption before entering a hypometabolic state (preferably a hibernation-like state), A program for determining the likelihood of death determines that a subject has died or is likely to have died, based at least on the subject's oxygen consumption, the carbon dioxide concentration in the subject's exhaled breath, the waveform of the subject's electrocardiogram, or the subject's blood pressure being zero. It may be a device that includes this.

[0022] (C') The apparatus of the present invention is as shown in Figure 12B, A calculation unit 205 calculates the oxygen consumption of the subject from the difference in oxygen concentration between the exhaled and inhaled air from the subject; A hibernation possibility determination unit 206 determines that the subject has entered a hibernation-like state, or is likely to have entered a hibernation-like state, based at least on a decrease in both oxygen consumption and theoretical setting temperature compared to the oxygen consumption and theoretical setting temperature of the subject in a non-hibernation-like state; and / or, A mortality determination unit 207 that determines whether a subject has died or is likely to have died, based at least on the oxygen consumption, the carbon dioxide concentration in the subject's exhaled breath, the waveform of the subject's electrocardiogram, or the subject's blood pressure being zero; The apparatus (C) may further include a calculation unit 220 for calculating the theoretical set temperature. The theoretical set temperature of body temperature (T R) is the outside temperature (or the ambient temperature of the subject) (T A While changing (for example, decreasing) core body temperature (T B Determine the relationship between ) and oxygen consumption (VO2), and determine the core body temperature (T) when oxygen consumption (VO2) is 0. B ) is obtained as an estimated value. Therefore, the above device (C) may further include a deep body thermometer 210 and a body temperature storage unit 210a that stores the body temperature measured by the thermometer. In the above device (C), the calculation unit 220 that calculates the theoretical set temperature determines the deep body temperature (T) when the oxygen consumption (VO2) is 0 from the relationship between the stored deep body temperature and the oxygen consumption. B The estimated value of core body temperature (T) can be calculated as the theoretical target temperature. B The relationship between (ΔVO2) and oxygen consumption (VO2) can be determined by curve fitting to an approximation curve, and in some embodiments, the approximation curve can be linear. Furthermore, according to the present invention, the feedback gain (H) of heat generation is given by H = ΔVO2 / ΔT B This can be determined as follows. Therefore, the apparatus (C) may further include a calculation unit 230 for calculating the heat generation feedback gain (H). The apparatus (C) may further include a determination unit 240 that takes the determinations from a hibernation possibility determination unit 206 and a mortality possibility determination unit 207, respectively, and determines whether the subject is in a hibernating state or a deceased state. The determination unit 240 is connected to an output device 250, and the result can be output from the output device 250. Therefore, in one embodiment, the apparatus (C) may further include a deep body thermometer, a body temperature storage unit for storing the body temperature measured by the thermometer, a calculation unit for calculating a theoretical set temperature, and a calculation unit for calculating the heat generation feedback gain (H). The apparatus (C) may further include an exhalation measurement unit 201 for measuring the oxygen concentration in exhaled air and an inhalation measurement unit 202 for measuring the oxygen concentration in inhaled air.

[0023] The apparatus of the present invention will be described in detail below. (First embodiment) In the first embodiment, the apparatus of the present invention has the configuration described in (A1) above. The apparatus of the present invention induces a hibernation-like state in a living subject by electrically stimulating the QRFP-producing neurons in the subject's brain. The first embodiment will be described below with reference to Figures 5 and 6.

[0024] The apparatus 1 of the present invention is A control unit 10 transmits a control signal to control the generation of voltage, A voltage generating unit 20 that receives a control signal from the control unit and generates a voltage, A stimulation probe having an electrical stimulation electrode distal to the voltage generating unit, wherein the stimulation probe 30 is long enough to access QRFP-producing neurons from the brain surface, and generates electrical stimulation at the distal electrical stimulation electrode 40 by the voltage from the voltage generating unit. It has.

[0025] In the apparatus 1 of the present invention, the control unit 10 transmits a control signal to control voltage generation. The control unit 10 may include control elements (microprocessor and power supply or battery). A single control signal can control one or more voltage generation events. Alternatively, the control signal can be transmitted multiple times to control multiple voltage generation events. The control signal can apply a single voltage stimulus, but may also control voltage generation to apply multiple stimuli until a hibernation-like state is induced in the subject {however, after the induction of the hibernation-like state, stimuli may or may not be applied}.

[0026] In the apparatus 1 of the present invention, the voltage generating unit 20 is electrically connected to the control unit 10 by wiring 15, and can generate a voltage by receiving a control signal from the control unit 10. The voltage can be, for example, 0 to 5 volts (V) and can be varied in increments of, for example, 0.1 volts. The voltage can be, for example, a pulse, with a pulse width of, for example, several tens of microseconds, and a stimulation frequency of, for example, several tens to several hundred pps. The voltage may start at, for example, 1 volt and be adjusted to increase until an effect is observed.

[0027] Although an example has been described in which the control unit 10 and the voltage generation unit 20 are connected by wiring 15, in the apparatus 1 of the present invention, instead of wiring 15, the control unit 10 and the voltage generation unit 20 may be able to communicate wirelessly between the control signal transmission unit 11 of the control unit 10 and the control signal reception unit 21 of the voltage generation unit, as shown in Figure 2. In this embodiment, the voltage generation unit 20 may have a battery 20a. The battery 20a may be rechargeable by a non-contact method. If it is rechargeable by a non-contact method, the battery 20a can be charged from outside the body even if it is located inside the body.

[0028] The voltage generating unit 20 transmits the voltage it generates to the stimulation probe 30 and the stimulation electrode 40 located at its tip via the extension cable 25. The distal end (i.e., tip) of the stimulation probe 30 has a stimulation electrode 40, which can apply voltage to brain tissue.

[0029] The stimulation probe 30 can be inserted into the brain by stereotactic brain surgery to accurately deliver the stimulation electrode 40 to the QRFP-producing neuron. Stereotactic brain surgery is a procedure in which the head is fixed with a measuring frame, and the electrode is inserted with an accuracy of 1 mm or less at the insertion position determined by CT scan or MRI. From the perspective of stereotactic brain surgery, the stimulation probe 30 is made of a material that is rigid enough not to bend or stretch when punctured deep into the brain (for example, a rigid material such as tungsten). The stimulation probe 30 is not particularly limited, but may have a diameter of approximately 1 μm to 1 mm, or 1 mm to 2.5 mm. The stimulation probe 30 has one or more (for example, two, three, or four) stimulation electrodes 40 distal to it. The stimulation electrode 40 can be approximately 1 to 5 mm in length along the long axis of the stimulation probe 30. When the stimulation probe 30 has multiple stimulation electrodes 40, the stimulation electrodes 40 may be arranged at intervals of approximately 1 mm to 1.5 mm, although this is not particularly limited. Each of the stimulating electrodes 40 may be controlled collectively by a single control signal, or, preferably, each may be controlled separately by individual control signals. By controlling each separately by individual control signals, it becomes possible to selectively generate voltage on the electrode that is optimal in relation to the electrode insertion position, thereby stimulating the brain.

[0030] The device 1 of the present invention induces a hibernation-like state in a subject and does not need to be portable. Here, "portable" means that it moves along with the subject as it moves relative to the ground where the subject is located (for example, the ground, or the floor of a vehicle if the subject is on a vehicle). Therefore, the device of the present invention may be fixed in place. The device of the present invention can be connected to a power source and therefore may not have batteries or rechargeable batteries. The device 1 of the present invention induces a hibernation-like state in a subject with a disease and does not need to be portable, but it is preferable to be portable from the viewpoint of treating the acute disease on the spot. The device of the present invention can be connected to a power source and therefore may not have batteries or rechargeable batteries, but from the viewpoint of portability, it may have batteries or rechargeable batteries so that it can operate without being connected to a power source.

[0031] (Second Embodiment) In the first embodiment, a device for electrically stimulating the deep brain was disclosed, while the second embodiment relates to a device for chemically stimulating the deep brain. The second embodiment will be described below with reference to Figure 7.

[0032] In a second embodiment, the apparatus 100 of the present invention is: A control unit 110 transmits a control signal that controls the release of QRFP-producing neuron-stimulating compounds, The storage section 125 for the compound, A compound dispensing unit 120 receives a control signal from the control unit and dispenses the compound from the compound storage unit 125, A guide 130 is provided with a compound release port 140 and a compound channel leading to the release port 140, for delivering the compound to the QRFP-producing neuron. The apparatus 100 of the present invention has the following features. In the apparatus 100 of the present invention, the control unit 110 is electrically connected to the compound delivery unit 120 via wiring 115. The compound delivery unit 120 receives a control signal from the control unit 110 and, in accordance with the control signal, releases the compound stored in the storage unit 125 from the storage unit 125 through the flow path 126 and flow path 121, and the guide 130 into the brain via the compound discharge port 140. The compound may be in the form of a solution dissolved in a solvent and can be delivered to the compound discharge port 140 by the liquid delivery mechanism of the compound delivery unit 120. The compound storage unit 125 may have a compound inlet 125a for introducing compounds from the outside. The compound inlet 125a can supply compounds to the compound storage unit. The compound storage unit 125 may be exposed outside the body. However, if the compound storage unit 125 is exposed outside the body, the compound storage unit 125 is maintained under sterile conditions. The control unit 110 transmits a control signal to the compound dispensing unit 120, for example, to dispense 1 μL to 100 μL of compound per dispensing cycle.

[0033] The guide 130 can be inserted into the brain by stereotactic surgery to accurately guide the compound release port 140 to the QRFP-producing neuron. Stereotactic surgery is a procedure in which the head is fixed with a measuring frame, and electrodes are inserted with an accuracy of 1 mm or less at the insertion position determined by CT scan or MRI. From the perspective of stereotactic surgery, the guide 130 is made of a material that is rigid enough not to bend or stretch when punctured deep into the brain (for example, a rigid material such as tungsten). The stimulation probe 30 may have a diameter of, for example, 1 mm to 2.5 mm.

[0034] The device 100 of the present invention induces a hibernation-like state in a subject and does not need to be portable. Here, "portable" means that it moves with the subject as it moves relative to the ground where the subject is located (e.g., the ground, or the floor of a vehicle if the subject is in a vehicle). Therefore, the device of the present invention may be fixed to its installation location (e.g., a bed on which the subject lies or the floor on which the bed is placed). The device of the present invention can be connected to a power source and therefore may not have batteries or rechargeable batteries. The device 100 of the present invention induces a hibernation-like state in a subject with a disease and does not need to be portable, but portability is preferable from the viewpoint of treating the disease on the spot if it is an acute disease. The device of the present invention can be connected to a power source and therefore may not have batteries or rechargeable batteries, but from the viewpoint of portability, it may have batteries or rechargeable batteries so that it can operate without being connected to a power source.

[0035] (Additional configuration) Apparatus 1 of the first embodiment and apparatus 100 of the second embodiment are (B) composition: The outside temperature gauge reads 50°C, A thermometer 60, A calculation unit 70 calculates the oxygen consumption of the subject from the difference in oxygen concentration between the exhaled and inhaled air from the subject, A recording unit 80 records the measured ambient temperature and at least one value selected from the group consisting of body temperature and oxygen consumption. It may further include {here, the thermometer may preferably be a core thermometer that measures the core body temperature of the subject}. The above (B) may be provided by the control unit 10 or control unit 110, for example, as shown in Figure 8 {here, although not shown in Figure 8, the control units 10 and 110 are connected to the voltage generation unit 20 by wire or wireless, as described in the first and second embodiments}. When inducing a hibernation-like state in the subject, the outside temperature (or the ambient temperature of the subject) (T A ) lowers the target's core body temperature (T B ) and metabolism are reduced. Therefore, by providing an ambient thermometer for measuring the ambient temperature (or the ambient temperature of the subject) and a body thermometer (preferably a core body thermometer), the device of the present invention can monitor the relationship between the subject's body temperature (preferably core body temperature) and the ambient temperature.

[0036] Apparatus 1 of the first embodiment and Apparatus 100 of the second embodiment have, in addition to the configuration of (B), configuration of (C). A calculation unit 205 calculates the oxygen consumption of the subject from the difference in oxygen concentration between the exhaled and inhaled air from the subject; A unit 206 for determining the possibility of a hypometabolic state (preferably a hibernation-like state) determines, at least based on a decrease in oxygen consumption compared to the oxygen consumption of the subject before entering a hypometabolic state (preferably a hibernation-like state), that the subject has entered or may have entered a hypometabolic state (preferably a hibernation-like state); and / or, A mortality determination unit 207 that determines whether a subject has died or is likely to have died, based at least on the oxygen consumption, the carbon dioxide concentration in the subject's exhaled breath, the waveform of the subject's electrocardiogram, or the subject's blood pressure being zero; The apparatus may further include the following. The apparatus having the configuration of (C) above can be used to determine whether a subject is in a hibernation-like state or has died when the subject has a serious illness. The apparatus (C) may further include a determination unit 240 that takes the determinations from a determination unit 206 for the possibility of a low metabolic state (preferably a hibernation-like state) and a determination unit 207 for the possibility of death, respectively, and determines whether the subject is in a hibernation-like state or has died. The determination unit 240 is connected to an output device 250, and the result can be output from the output device 250. Therefore, the apparatus (C) may further include an exhalation measurement unit 201 for measuring the oxygen concentration in exhaled air and an inhalation measurement unit 202 for measuring the oxygen concentration in inhaled air.

[0037] Apparatus 1 of the first embodiment and Apparatus 100 of the second embodiment have, in addition to the configuration of (B), the configuration of (C): A calculation unit that calculates the oxygen consumption of the subject from the difference in oxygen concentration between the subject's exhaled and inhaled air; A hibernation possibility determination unit that determines that an object has entered a hibernation-like state, or is likely to have entered a hibernation-like state, based at least on a decrease in both oxygen consumption and theoretical setting temperature compared to the oxygen consumption and theoretical setting temperature of an object in a non-hibernation-like state; and / or, A mortality determination unit that determines whether a subject has died or is likely to have died, based at least on the oxygen consumption, the carbon dioxide concentration in the subject's exhaled breath, the waveform of the subject's electrocardiogram, or the subject's blood pressure being zero; The device may further include the following. The device having the configuration of (C) above can be used to determine whether a subject is in a hibernation-like state or has died when the subject has a serious illness. The device having the configuration of (C) above may further include a deep body thermometer and a body temperature storage unit that stores the body temperature measured by the thermometer. The device having the configuration of (C) above may further include a calculation unit that calculates a theoretical set temperature. The device having the configuration of (C) above may further include a calculation unit that calculates a heat generation feedback gain (H). The device having the configuration of (C) above may further include a deep body thermometer, a body temperature storage unit that stores the body temperature measured by the thermometer, a calculation unit that calculates a theoretical set temperature, and a calculation unit that calculates a heat generation feedback gain (H). The hibernation possibility determination unit may determine that a subject has entered a hibernation-like state or may have entered a hibernation-like state, at least based on the fact that the oxygen consumption, theoretical set temperature, and heat generation feedback gain have all decreased compared to the oxygen consumption, theoretical set temperature, and heat generation feedback gain of a subject in a non-hibernation-like state.

[0038] Furthermore, the apparatus of the present invention includes an exhaled gas analysis unit 70 that measures the oxygen concentration in the exhaled gas, thereby enabling the estimation of the oxygen consumption (VO2) of the subject, and the estimation of the subject's metabolic state from the oxygen consumption (VO2).

[0039] Also, core body temperature (T B From ) and oxygen consumption (VO2), the theoretical set temperature of body temperature (T R It also becomes possible to estimate the theoretical set temperature of body temperature (T). R ) is the outside temperature (or the ambient temperature of the subject) (T A While changing (for example, decreasing) core body temperature (T B Determine the relationship between ) and oxygen consumption (VO2), and determine the core body temperature (T) when oxygen consumption (VO2) is 0. B It is calculated as an estimate of core body temperature (T). BThe relationship between the heat generation rate and oxygen consumption (VO2) can be determined, for example, by linear regression. Furthermore, the feedback gain (H) for heat generation is given by H = ΔVO2 / ΔT B It can be calculated as follows: Here, ΔVO2 is the change in VO2, and ΔT B is T B This is a change.

[0040] The apparatus of the present invention may further include a recording unit 80 that records the measured ambient temperature and at least one numerical value selected from the group consisting of body temperature (preferably core body temperature) and oxygen concentration. The apparatus of the present invention may further include an oxygen consumption determination unit 90 that determines the oxygen consumption of the target from the oxygen concentration in the exhaled gas. The apparatus of the present invention may further include a theoretical set temperature (T) for body temperature. R The apparatus of the present invention may further include an estimation unit 91 that estimates the theoretical set temperature (T) of body temperature and the feedback gain (H) of heat generation. R The apparatus may further include a determination unit 92 that determines whether the subject has been induced into a hibernation-like state based at least on the feedback gain (H) of heat generation and / or whether the subject has died based at least on the oxygen consumption becoming zero. The determination unit for hibernation possibility and the determination unit for death possibility described above may be the same, but may also be provided separately as separate components within the apparatus. The apparatus of the present invention may further include an output unit 93 for information on whether a hibernation-like state has been induced and / or whether the subject has died. Examples of the output unit 93 include a display that shows the information and / or a printer that prints the information. Information on whether a hibernation-like state has been induced may include information that a hibernation-like state has been induced and information that a hibernation-like state has not been induced, and can be output by the output unit 93. Information on whether the subject has died may include information that the subject has died and information that the subject has not died, and can be output by the output unit 93.

[0041] (Third embodiment) According to the present invention, A device for determining hibernation, In mammals such as humans in which the test compound was administered to the anterior ventral periventricular nucleus (AVPe), medial preoptic area (MPA), and periventricular nucleus (Pe), at least two different ambient temperatures (T) were observed before and after administration. A Oxygen consumption (VO2) and core body temperature (T) recorded under the following conditions. B A recording unit that records ) and The system includes a calculation unit that estimates the correlation between oxygen consumption and core body temperature before and after administration, determines from the estimated correlation whether the degree of decrease in oxygen consumption when core body temperature decreases decreases after administration compared to before administration, and determines whether the estimated core body temperature, assuming oxygen consumption is 0, decreases after administration compared to before administration. A determination unit determines that the mammal has entered hibernation based at least on the fact that the degree of decrease in oxygen consumption when core body temperature decreases is lower after administration compared to before administration, and the estimated core body temperature assuming oxygen consumption is 0 is lower after administration compared to before administration. ferrous device A is provided. In one preferred embodiment, the subject has a disease. In one preferred embodiment, the subject has a chronic disease. In one preferred embodiment, the subject has an acute disease. In one preferred embodiment, the disease, chronic disease, or acute disease is a serious disease. The acute disease may be one or more selected from the group consisting of, for example, sepsis, acute renal failure, acute pneumonia, myocardial infarction, cerebral infarction, cerebral hemorrhage, and hemorrhagic shock. The chronic disease may be one or more selected from the group consisting of tumors, autoimmune diseases, and aging.

[0042] The recording unit records at least two different ambient temperatures (T A Oxygen consumption (VO2) and core body temperature (T) recorded under the following conditions. B ) records. The recording unit records one T A For one VO2 and T B The recorded oxygen consumption (VO2) and core body temperature (T) are stored in association with each other. BThe data is read from the recording unit and transmitted to the calculation unit, where the correlation between oxygen consumption and core body temperature is estimated. In one embodiment, the correlation is linear. After the correlation is estimated, the calculation unit determines whether the degree of decrease in oxygen consumption when core body temperature decreases is lower after administration compared to before administration, and whether the estimated core body temperature, assuming oxygen consumption is 0, is lower after administration compared to before administration. Based on the determination in the calculation unit, the determination unit can determine that the mammal has entered hibernation if the degree of decrease in oxygen consumption when core body temperature decreases is lower after administration compared to before administration, and the estimated core body temperature, assuming oxygen consumption is 0, is lower after administration compared to before administration. The determination unit may determine that the mammal did not hibernate (or that it did not hibernate) if the degree of decrease in oxygen consumption when core body temperature decreases does not decrease after administration compared to before administration, or if the estimated core body temperature, assuming oxygen consumption is 0, does not decrease after administration compared to before administration.

[0043] The apparatus for determining hibernation in the third embodiment of the present invention may further include a deep body thermometer and an exhaled gas analysis unit for measuring the oxygen concentration in exhaled gas. The apparatus in the third embodiment may further include an output unit that receives hibernation determination information from the determination unit and outputs the information. The information output unit may be a user interface such as a display, a recording device for non-volatile memory such as a USB memory and an SD card, an information transmission device for wireless communication to an external source, or a printing device for a medium such as paper such as a printer.

[0044] The apparatus of the first or second embodiment may further include the apparatus for determining hibernation in the third embodiment.

[0045] (Stimulation method of the present invention) The present invention provides a method for reducing the theoretical set temperature of body temperature and / or the feedback gain of heat generation in a subject. The present invention provides a method for inducing a hibernation-like state in a subject. In one preferred embodiment, the subject has a disease. In one preferred embodiment, the subject has a chronic disease. In one preferred embodiment, the subject has an acute disease. In one preferred embodiment, the disease, chronic disease or acute disease is a serious disease. An acute disease may be one or more selected from the group consisting of, for example, sepsis, acute renal failure, acute pneumonia, myocardial infarction, cerebral infarction, cerebral hemorrhage, and hemorrhagic shock. A chronic disease may be one or more selected from the group consisting of tumors, autoimmune diseases, and aging. The present invention provides a method for reducing the feedback gain of thermogeneration in a subject, comprising providing excitatory stimulation to pyroglutaminated RF amide peptide (QRFP)-producing neurons. The present invention also provides a method for reducing the feedback gain of thermogeneration in a subject, comprising providing excitatory stimulation to pyroglutaminated RF amide peptide (QRFP)-producing neurons. The present invention also provides a method for reducing the theoretical set temperature of body temperature and the feedback gain of thermogeneration in a subject, comprising providing excitatory stimulation to pyroglutaminated RF amide peptide (QRFP)-producing neurons. The present invention also provides a method for inducing a hibernation-like state in a subject, comprising providing excitatory stimulation to pyroglutaminated RF amide peptide (QRFP)-producing neurons using a drug or the like. In a preferred embodiment, the subject has a disease. In a preferred embodiment, the subject has a chronic disease. In a preferred embodiment, the subject has an acute disease. In a preferred embodiment, the disease, chronic disease or acute disease is a serious disease. Acute diseases may include one or more selected from the group consisting of, for example, sepsis, acute renal failure, acute pneumonia, myocardial infarction, cerebral infarction, cerebral hemorrhage, and hemorrhagic shock. Chronic diseases may include one or more selected from the group consisting of tumors, autoimmune diseases, and aging.

[0046] In the method of the present invention, target pyroglutaminated RF amido peptide (QRFP)-producing neurons can be stimulated, for example, using the apparatus of the present invention. In the method of the present invention, QRFP-producing neurons can be stimulated by applying a voltage to them. In the method of the present invention, QRFP-producing neurons can also be stimulated by specifically expressing a receptor (e.g., hM3Dq) in pyroglutaminated RF amido peptide (QRFP)-producing neurons, for example, using the DREADD method, and then administering a ligand (e.g., clozapine-N-oxide (CNO)) to the receptor. hM3Dq can be expressed in target QRFP-producing neurons by infecting them with a virus (e.g., adenovirus, adeno-associated virus, etc.) having a gene encoding hM3Dq operably linked to a QRFP promoter. CNO can be administered to the brain, for example, using the apparatus of the present invention. In one preferred embodiment, the subject has a disease. In one preferred embodiment, the subject has a chronic disease. In one preferred embodiment, the subject has an acute disease. In a preferred embodiment, a disease, chronic disease, or acute disease is a serious illness. An acute disease may be one or more selected from the group consisting of, for example, sepsis, acute renal failure, acute pneumonia, myocardial infarction, cerebral infarction, cerebral hemorrhage, and hemorrhagic shock. A chronic disease may be one or more selected from the group consisting of tumors, autoimmune diseases, and aging.

[0047] In the method of the present invention, in the subject, pyroglutaminated RF amidopeptide (QRFP)-producing neurons can also be stimulated with an activator for these neurons. Activators can be screened using QRFP neurons or explored using cultured cells in which receptors expressed on QRFP neurons are forced to express. The neuronal activator may be administered locally to the QRFP-producing neurons using an applicator. QRFP-producing neuron-specific activators may be administered by intracerebroventricular, intrathecal, and systemic administration such as intravenous administration. In one preferred embodiment, the subject has a disease. In one preferred embodiment, the subject has a chronic disease. In one preferred embodiment, the subject has an acute disease. In one preferred embodiment, the disease, chronic disease, or acute disease is a serious disease. An acute disease may be one or more selected from the group consisting of, for example, sepsis, acute renal failure, acute pneumonia, myocardial infarction, cerebral infarction, cerebral hemorrhage, and hemorrhagic shock. A chronic disease may be one or more selected from the group consisting of tumors, autoimmune diseases, and aging.

[0048] The method of the present invention may further include lowering the ambient temperature. This will reduce the target T B This can reduce T in a hibernation-like state. B When this decreases, a low metabolic state occurs, which is thought to reduce energy consumption and allow life to be sustained.

[0049] The method of the present invention relates to the target core body temperature (T B The method of the present invention may further include measuring the oxygen concentration of the exhaled breath of the subject.

[0050] The method of the present invention may further include estimating the oxygen consumption (VO2) of a subject. The oxygen consumption (VO2) of a subject can be estimated, for example, from the difference in oxygen concentration between inhalation and exhalation.

[0051] The present invention provides (or records) oxygen consumption and core body temperature recorded under at least two different ambient temperature conditions before and after administration in mammals such as humans in which the test compound is administered to the regions of the anterior ventral periventricular nucleus (AVPe), medial preoptic area (MPA), and periventricular nucleus (Pe), respectively. To estimate the correlation between oxygen consumption and core body temperature both before and after administration, This includes determining, based on the estimated correlation, whether the degree of decrease in oxygen consumption when core body temperature decreases decreases after administration compared to before administration, and whether the estimated core body temperature, assuming oxygen consumption is 0, decreases after administration compared to before administration. A possible method for indicating that a mammal has hibernated is when the degree of decrease in oxygen consumption when core body temperature decreases decreases after administration compared to before administration, and the estimated core body temperature, assuming oxygen consumption is 0, decreases after administration compared to before administration. In one preferred embodiment, the mammal has a disease. In one preferred embodiment, the mammal has a chronic disease. In one preferred embodiment, the mammal has an acute disease. In one preferred embodiment, the disease, chronic disease, or acute disease is a serious disease. An acute disease may be one or more selected from the group consisting of, for example, sepsis, acute renal failure, acute pneumonia, myocardial infarction, cerebral infarction, cerebral hemorrhage, and hemorrhagic shock. A chronic disease may be one or more selected from the group consisting of tumors, autoimmune diseases, and aging. In this embodiment, the method of the present invention relates to the theoretical set temperature (T) of the target body temperature. R This may further include estimating the theoretical set temperature (T R ) is the outside temperature (or the ambient temperature of the subject) (T A While changing (for example, decreasing) core body temperature (T B Determine the relationship between ) and oxygen consumption (VO2), and determine the core body temperature (T) when oxygen consumption (VO2) is 0. B It is calculated as an estimate of core body temperature (T). BThe relationship between ) and oxygen consumption (VO2) can be determined, for example, by linear regression.

[0052] The method of the present invention may further include estimating the feedback gain (H) of the heat generation of the subject. The feedback gain (H) of heat generation is given by H = ΔVO2 / ΔT B It can be calculated as follows.

[0053] The method of the present invention may further include determining whether the subject is in a hibernation-like state. Whether the subject is in a hibernation-like state is determined by the theoretical set temperature of the body temperature (T) when the ambient temperature is lowered. R This can be determined by whether both the feedback gain (H) for body temperature and the feedback gain for heat generation decrease. When the ambient temperature is lowered, the theoretical set temperature (T) of body temperature decreases. R If both the feedback gain (H) for ) and the feedback gain for heat generation decrease, it can be determined that the subject is in a hibernation-like state. A hibernation-like state may be beneficial in that it improves life-protective functions by reducing the body's metabolism. In one preferred embodiment, the subject has a disease. In one preferred embodiment, the subject has a chronic disease. In one preferred embodiment, the subject has an acute disease. In one preferred embodiment, the disease, chronic disease, or acute disease is a serious disease. An acute disease may be one or more selected from the group consisting of, for example, sepsis, acute renal failure, acute pneumonia, myocardial infarction, cerebral infarction, cerebral hemorrhage, and hemorrhagic shock. A chronic disease may be one or more selected from the group consisting of tumors, autoimmune diseases, and aging.

[0054] (The screening system of the present invention) According to the present invention, a method for screening substances that provide excitatory stimulation to pyroglutaminated RF amide peptide (QRFP) producing neurons located in the regions of the anterior ventral periventricular nucleus (AVPe), medial preoptic area (MPA), and periventricular nucleus (Pe), The test compound is brought into contact with the isolated QRFP-producing neurons, Measuring the excitation of the QRFP-producing neurons, Select a test compound that provides excitatory stimulation to the QRFP-producing neurons, A method is provided that includes the following. The method may be an in vitro method. Screening may be performed on subjects. In one preferred embodiment, subjects have a disease. In one preferred embodiment, subjects have a chronic disease. In one preferred embodiment, subjects have an acute disease. In one preferred embodiment, the disease, chronic disease or acute disease is a serious disease. In one preferred embodiment, subjects are disease models. The acute disease may be one or more selected from the group consisting of, for example, sepsis, acute renal failure, acute pneumonia, myocardial infarction, cerebral infarction, cerebral hemorrhage, and hemorrhagic shock. The chronic disease may be one or more selected from the group consisting of tumors, autoimmune diseases, and aging.

[0055] The excitation of QRFP-producing neurons can be measured electrically. Electrical measurement of neuronal excitation can be performed, for example, using conventional electrophysiological methods with depolarization of the membrane potential as an indicator. The membrane potential can be measured, for example, by nerve recording methods such as the microelectrode method or the patch-clamp method, or it may be measured using a fluorescent probe for membrane potential measurement. While not particularly limited, examples of fluorescent probes for membrane potential measurement include 4-(4-(didecylamino)styryl)-N-methylpyridinium iodide (4-Di-10-ASP), bis-(1,3-dibutylbarbiturate trimethine oxonol (DiSBAC2(3)), 3,3'-dipropylthiadicarbocyanine iodide (DiSC3(5)), 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1), and rhodamine 123. It can be cited as such. Furthermore, neuronal excitation can also be measured chemically. When a neuron is excited, the intracellular calcium concentration increases. For example, neuronal excitation can be measured using a calcium concentration indicator. Various probes are known as calcium concentration indicators, such as 1-[6-amino-2-(5-carboxy-2-oxazolyl)-5-benzofuranyloxy]-2-(2-amino-5-methylphenoxy)ethane-N,N,N',N'-tetraacetic acid, pentaacetoxymethyl ester (Fura 2-AM), and can be used in the present invention.

[0056] QRFP-producing neurons are neurons located within the anterior ventricular nucleus (AVPe), medial preoptic area (MPA), and periventricular nucleus (Pe), and can be classified as neuronal strains. Neuronal strains can be obtained by selecting strains in which neurons produce QRFP. Whether or not a neuron produces QRFP can be confirmed by conventional methods using an antibody against QRFP.

[0057] (Method for determining hibernation according to the present invention) The present invention's method for determining hibernation involves analyzing the effects of a drug that induces hibernation, is expected to induce hibernation, or has the potential to induce hibernation in a subject. If the subject enters a hibernation-like state, it can be maintained or terminated. If the subject does not enter a hibernation-like state, further treatment can be administered or treatment can be discontinued. In one preferred embodiment, the subject has a disease. In one preferred embodiment, the subject has a chronic disease. In one preferred embodiment, the subject has an acute disease. In one preferred embodiment, the disease, chronic disease, or acute disease is a serious disease. An acute disease may be one or more selected from the group consisting of, for example, sepsis, acute renal failure, acute pneumonia, myocardial infarction, cerebral infarction, cerebral hemorrhage, and hemorrhagic shock. A chronic disease may be one or more selected from the group consisting of tumors, autoimmune diseases, and aging. The method for determining hibernation according to the present invention may be a computational science method. The method for determining hibernation according to the present invention may not involve medical procedures. The method for determining hibernation according to the present invention may, for example, not involve measurement itself, but may determine based on input of measured values. The determination result may be basic information for diagnosis by medical professionals, including physicians. It can be difficult to quickly distinguish between a hibernation-like state and death based solely on external appearance. In certain embodiments, the hibernation determination method of the present invention can be used to distinguish between hibernation and death. If the subject is found to be in a death state, medical personnel can quickly initiate resuscitation or facilitate recovery from the hibernation-like state.

[0058] The method for determining hibernation according to the present invention is: A method for determining (by testing, prediction, estimation, or computational science) whether or not a test compound induces hibernation in mammals such as humans, or the possibility thereof. In mammals such as humans in which the test compound was administered to the anterior ventral periventricular nucleus (AVPe), medial preoptic area (MPA), and periventricular nucleus (Pe), the following data are provided (or recorded): oxygen consumption and core body temperature recorded under at least two different ambient temperature conditions before and after administration. To estimate the correlation between oxygen consumption and core body temperature both before and after administration, This includes determining, based on the estimated correlation, whether the degree of decrease in oxygen consumption when core body temperature decreases decreases after administration compared to before administration, and whether the estimated core body temperature, assuming oxygen consumption is 0, decreases after administration compared to before administration. A method to indicate that a mammal has entered hibernation is one in which the degree of decrease in oxygen consumption when core body temperature decreases decreases after administration compared to before administration, and the estimated core body temperature, assuming oxygen consumption is 0, decreases after administration compared to before administration. It is possible. Mammals may be non-human mammals. In a preferred embodiment, mammals have diseases. In a preferred embodiment, mammals have chronic diseases. In a preferred embodiment, mammals have acute diseases. In a preferred embodiment, a disease, chronic disease or acute disease is a serious disease. An acute disease may be one or more selected from the group consisting of, for example, sepsis, acute renal failure, acute pneumonia, myocardial infarction, cerebral infarction, cerebral hemorrhage, and hemorrhagic shock. A chronic disease may be one or more selected from the group consisting of tumors, autoimmune diseases, and aging.

[0059] Setting different ambient temperature conditions can be done by the temperature control unit (for example, the apparatus of the first or second embodiment). Oxygen consumption and core body temperature can be determined by an exhaled gas analyzer and a core body thermometer, respectively. The exhaled gas analyzer and core body thermometer can be those provided in the apparatus of the first or second embodiment.

[0060] (Treatment of disease by hypometabolism therapy (preferably by inducing hibernation)) The present invention provides a method for treating a subject or disease, comprising inducing a hypometabolic state (preferably a hibernation-like state) in the subject having the disease. The hypometabolic state can halt or delay the progression of symptoms of any disease. The hypometabolic state can be induced, for example, by hypometabolic therapy. Hibernation puts the subject into a hypothermic and hypometabolic state, while simultaneously halting or delaying the progression of symptoms of any disease. This can slow the progression of chronic diseases or, in the case of acute diseases, allow time to be secured before further treatment, for example, time for medical transport. Furthermore, by inducing a hypometabolic state (preferably a hibernation-like state), it is possible to maintain the patient's life by delaying the progression of the disease. In the subject, hibernation can be induced by exciting QRFP neurons. For example, in the subject, hibernation can be induced by the above method. In the subject, hibernation can be induced using the above device. The disease may be, for example, an acute disease. The acute disease may be one or more selected from the group consisting of, for example, sepsis, acute renal failure, acute pneumonia, myocardial infarction, cerebral infarction, cerebral hemorrhage, and hemorrhagic shock. The acute disease is preferably one in which blood circulation is preserved, with respect to promoting the delivery of the administered compound to the QRFP nerves. The disease may be, for example, a chronic disease or condition. The chronic disease may be one or more selected from the group consisting of tumors, autoimmune diseases, and aging.

[0061] (Compounds or candidates for inducing hibernation to treat diseases) According to the present invention, a compound or candidate for a hibernation-inducing compound can be obtained by administering the compound to a subject and then testing whether or not it induces hibernation in the subject. Accordingly, the present invention provides a method for selecting a compound or candidate for a hibernation-inducing compound, comprising administering the compound to a subject, then testing whether or not it induces hibernation in the subject, and selecting a compound that induces hibernation. The present invention also provides a method for testing a compound or a composition containing the compound (e.g., a pharmaceutical preparation and a pharmaceutical composition), comprising: administering the compound or composition to a subject; determining whether or not it induces hibernation in the subject; and obtaining a compound or composition that induces hibernation. The obtained compound can then be formulated as a pharmaceutical composition, or the obtained compound can be included in a composition formulated as a pharmaceutical preparation. The subject may be a subject with a disease, particularly a subject with a serious disease, for example, a patient with a serious acute or chronic disease. Examples of diseases may be as described above. The present invention provides a pharmaceutical composition containing such a selected compound. The pharmaceutical composition can be used to induce hibernation in the treatment of a disease.

[0062] (Device for treating diseases caused by hibernation) The present invention provides a device for treating diseases caused by hibernation. According to the present invention, for example, the device for treating diseases caused by hibernation may be the device of the first embodiment, the second embodiment, or the third embodiment described above. In this embodiment, the device for treating diseases caused by hibernation may further include a determination unit that estimates or determines that the subject has died or may have died, at least based on the fact that the oxygen consumption is zero (for example, when the oxygen consumption of the subject disappears).

[0063] According to the present invention, for example, a device for treating diseases caused by hibernation is mounted on an automobile. Therefore, according to the present invention, an automobile equipped with a device for treating diseases caused by hibernation is provided. The automobile may be an emergency transport vehicle (e.g., an ambulance). The automobile may be equipped with a bed for laying the patient. The automobile may be capable of carrying a stretcher. The automobile may be equipped with a fixing device on the floor of the interior for securing the stretcher. The fixing device may be an anti-vibration stand. The stretcher may be a leg-folding stretcher. The ambient temperature of the target can be adjusted by air conditioning or the like to be approximately the same as the theoretical set temperature (e.g., ±5°C, ±4°C, ±2°C, or ±1°C of the theoretical set temperature). According to the present invention, for example, a device for treating diseases caused by hibernation may be installed in a medical institution or a non-medical institution, preferably a medical institution. Among medical institutions, it may be preferably installed in a high care unit (HCU) and an intensive care unit (ICU). The ambient temperature of the target can be adjusted by air conditioning or other means to approximately the same as the theoretical set temperature (for example, ±5°C, ±4°C, ±2°C, or ±1°C from the theoretical set temperature).

[0064] (Monitoring device for low metabolic state) In certain cases, such as when the disease is severe, it is beneficial to monitor minute oxygen consumption during a hypometabolic state (preferably a hibernation-like state) to confirm the patient's survival. Oxygen consumption can be monitored, for example, by measuring the oxygen concentration in both inhaled and exhaled breath and comparing the exhaled and inhaled measurements. When a patient enters a hypometabolic state (preferably a hibernation-like state), oxygen consumption decreases compared to before the state, and the difference between the exhaled and inhaled oxygen concentration measurements becomes smaller. However, when the patient dies, the difference between the exhaled and inhaled oxygen concentration measurements disappears. By monitoring the patient's respiration in this way, the patient's condition (for example, a patient with a serious disease or condition, e.g., a patient with a serious acute or chronic disease) can be monitored.

[0065] The present invention provides a device that enables monitoring of a patient's hypometabolic state (preferably a hibernation-like state) and survival status based on oxygen consumption. According to the present invention, as described above, when a patient enters a hypometabolic state (preferably a hibernation-like state), oxygen consumption decreases compared to before the hypometabolic state (preferably a hibernation-like state), and the difference between the measured oxygen concentrations in exhaled and inhaled air becomes smaller. When the patient dies, the difference between the measured oxygen concentrations in exhaled and inhaled air disappears. Therefore, the present invention provides a device for monitoring a hypometabolic state (preferably a hibernation-like state) in a patient with a disease, A calculation unit that calculates the oxygen consumption of the subject from the difference in oxygen concentration between the subject's exhaled and inhaled breath, A unit for estimating the possibility of a hypometabolic state (preferably a hibernation-like state) estimates that the subject has entered a hypometabolic state (preferably a hibernation-like state), or is likely to have entered one, based at least on a decrease in oxygen consumption compared to the subject's oxygen consumption before entering a hypometabolic state (preferably a hibernation-like state), A mortality probability estimation unit that estimates that the subject has died or may have died, at least based on the fact that oxygen consumption is zero. including, device The present invention provides a device for monitoring a hibernation-like state in a subject with a disease that is in a hibernation-like state, A calculation unit that calculates the oxygen consumption of the subject from the difference in oxygen concentration between the subject's exhaled and inhaled breath, A unit for estimating the possibility of hibernation estimates that the subject has entered a hibernation-like state, or is likely to have entered a hibernation-like state, based at least on a decrease in oxygen consumption compared to the subject's oxygen consumption before entering a hibernation-like state. A mortality probability estimation unit that estimates that the subject has died or may have died, at least based on the fact that oxygen consumption is zero. including, device The present invention provides a device for monitoring a hibernation-like state in a subject with a disease that is in a hibernation-like state, A calculation unit that calculates the oxygen consumption of the subject from the difference in oxygen concentration between the subject's exhaled and inhaled breath, A unit for estimating the possibility of hibernation estimates that the subject has entered a hibernation-like state, or is likely to have entered a hibernation-like state, based at least on a decrease in both oxygen consumption and theoretical temperature compared to the oxygen consumption and theoretical temperature of the subject in a non-hibernation-like state, A mortality probability estimation unit that estimates that the subject has died or may have died, at least based on the fact that oxygen consumption is zero. including, device It will be provided.

[0066] Furthermore, the present invention provides a device for monitoring a hypometabolic state (preferably a hibernation-like state) in a subject with a disease that is in a hypometabolic state (preferably a hibernation-like state), comprising: an exhalation measurement unit that measures the oxygen concentration of the subject's exhaled breath over time; an inhalation measurement unit that measures the oxygen concentration of the subject's inhaled breath over time; a calculation unit (e.g., a processor) that calculates the amount of oxygen consumption over time from the obtained oxygen concentrations of the inhaled and exhaled breath at the same time; a memory (e.g., a volatile memory or a non-volatile memory) that stores the oxygen consumption calculated over time; and a state determination unit (e.g., a processor) that determines, at least based on the change in oxygen consumption over time (preferably also based on a decrease in the subject's theoretical set temperature), that the subject may have entered a hypometabolic state (preferably a hibernation-like state) or may have died. Furthermore, the present invention provides a device for monitoring a hibernation-like state in a subject with a disease, comprising: an exhalation measurement unit that measures the oxygen concentration of the subject's exhaled breath over time; an inhalation measurement unit that measures the oxygen concentration of the subject's inhaled breath over time; a calculation unit (e.g., a processor) that calculates the amount of oxygen consumption over time from the obtained oxygen concentrations of the inhaled and exhaled breath at the same time; a memory (e.g., a volatile memory or a non-volatile memory) that stores the oxygen consumption calculated over time; and a state determination unit (e.g., a processor) that determines whether the subject may have entered a hibernation state or may have died, based at least on the change in the amount of oxygen consumption over time (preferably also based on a decrease in the subject's theoretical set temperature). The calculation unit and the state determination unit may be implemented in the same processor. In one embodiment, the oxygen consumption monitoring device may further have an output unit (e.g., a display) that outputs the amount of oxygen consumption or its change over time. In one embodiment, the calculation unit calculates a graph including the change in oxygen consumption over time, and the output unit outputs the graph. The graph may, for example, have oxygen consumption on the vertical axis and time on the horizontal axis. In this embodiment, the output unit may output a graph showing the change in oxygen consumption over time and the possibility of the subject being in a state of hibernation and / or death. The output unit may, for example, be a display that can show a graph showing the change in oxygen consumption over time and the possibility of the subject being in a state of hibernation and / or death.If a person may be in a state of death, a doctor or medical professional can assess the situation and, if necessary, provide first aid, treatment, or resuscitation.

[0067] According to the present invention, a device for monitoring a patient's condition by measuring oxygen consumption (the hibernation monitoring device of the present invention) can be incorporated into, linked to, or operated in conjunction with a device for determining hibernation according to a third embodiment of the present invention. The present invention provides a hibernation monitoring device of the present invention incorporated into, linked to, or operated in conjunction with a device for determining hibernation according to a third embodiment of the present invention. In one embodiment, according to the present invention, the oxygen consumption monitoring device may further include a receiving unit that receives a theoretical set temperature of an object obtained from a device for determining hibernation according to a third embodiment of the present invention, and may further include a state determination unit (e.g., a processor) that determines whether the object may have entered a hibernation state or may have died, based at least on the information received by the receiving unit and information on the oxygen consumption or its change over time. This may improve the accuracy of hibernation determination. It may also enable a more accurate understanding of the condition of a patient with an acute illness.

[0068] The present invention provides a device that enables monitoring of a patient's hypometabolic state (preferably a hibernation-like state) and survival status based on oxygen consumption, a program that causes a processor to perform such monitoring, a method for causing a processor to perform such monitoring, and a method and program for controlling the device. The method for controlling the device that enables monitoring of a patient's hypometabolic state (preferably a hibernation-like state) and survival status based on oxygen consumption includes acquiring the oxygen concentration in the subject's exhaled breath (e.g., in an exhaled breath measurement unit), acquiring the oxygen concentration in the subject's inhaled breath (e.g., in an inhaled breath measurement unit), and estimating or determining the subject's oxygen consumption at at least several time points (e.g., in a calculation unit, e.g., a processor) from the acquired oxygen concentrations in the inhaled and exhaled breath. In this embodiment, it may further include outputting the oxygen consumption or its change over time. Whether or not the oxygen consumption is lower than normal can be used by healthcare professionals, including physicians, as information to determine whether or not the subject is in a hypometabolic state (preferably a hibernation-like state). Furthermore, this embodiment may further include determining (for example, in a state determination unit, e.g., a processor) whether the subject is in a hypometabolic state (preferably a hibernation-like state) and / or has died, based on the estimated or determined oxygen consumption, and outputting the results (for example, by a display device such as a display). Healthcare professionals, including physicians, can use the output results as information to determine whether the subject is in a hibernation state and / or has died.

[0069] According to the present invention, a program is provided for causing the apparatus of the present invention to perform this method.

[0070] (Methods to induce a hypometabolic state in peripheral tissues) The present invention provides a method for inducing a hypometabolic state in a mammal. The present invention provides a method for inducing a hypometabolic state in peripheral tissues in a diseased mammal. The present invention provides a method for inducing a hypometabolic state in peripheral tissues in a diseased mammal, comprising inducing a hypometabolic state in the mammal, thereby inducing a hypometabolic state in the peripheral tissues. In some embodiments, the mammal may be non-human.

[0071] The present invention provides a method for treating a mammal having a disease (or the disease in said mammal), the method comprising inducing a hypometabolic state in said mammal. According to the present invention, inducing a hypometabolic state in said mammal can reduce the rate of disease progression in said mammal. According to the present invention, inducing a hypometabolic state in said mammal can also extend the survival period (or time to death) in said mammal. According to the present invention, inducing a hypometabolic state in said mammal can also reduce the probability of death in said mammal.

[0072] According to the present invention, a hypometabolic state can be induced by hypometabolic therapy. According to the present invention, a hypometabolic state can be a hibernation-like state. A hibernation-like state can be induced by the method according to the present invention. For example, a hibernation-like state can be induced by stimulating QRFP neurons. Stimulation of QRFP neurons can be performed using the apparatus of the present invention.

[0073] Mammals in a low metabolic state may be monitored. This monitoring can be carried out by monitoring changes in oxygen consumption over time.

[0074] According to the present invention, the treatment may be emergency treatment or life-sustaining treatment. According to the present invention, for example, the treatment may be performed during transport of the animal to a medical institution (e.g., a hospital) (e.g., during emergency transport) or in the intensive care unit of the medical institution.

[0075] According to the present invention, a method for testing the effectiveness of a treatment, Using a diseased mammal for the procedure, Observe the treated mammals and Compared to a control group that did not receive the treatment, it is determined whether the treatment reduces the rate of disease progression, reduces mortality, or extends survival time. A method is provided which includes determining whether a treatment is effective based on the fact that, compared to a control group that did not receive the treatment, the treatment reduces the rate of disease progression, reduces mortality, and extends survival. The treatment may be, for example, a treatment that induces a hypometabolic state (preferably a hibernation-like state), in which case the treatment may be shown to be effective. [Examples]

[0076] [Experimental Methods] (1)Animals All animal experiments were conducted at the International Institute of Integrated Sleep Medicine (IIIS), the University of Tsukuba, and the RIKEN Biosystems Dynamics Research Center (BDR), in accordance with animal experiment guidelines. Approval was obtained from the animal experiment committees of each institution, and the NIH guidelines were followed. Except for the dormancy induction experiments, mice were given free access to food and water. A The environment was maintained at 22°C, 50% relative humidity, and a 12-hour light / 12-hour dark cycle. Mice weighing 34g or more were found not to exhibit reproducible FIT (Fitness Inhibitory Time), so heavier mice weighing 34g or more were excluded from the diapause experiment.

[0077] Qrfp-iCre mice were generated by homologous recombination in C57BL / 6N embryonic stem cells and transplantation in 8-cell stage embryos (ICRs). The targeted vector was designed so that the endogenous Qrfp promoter would promote iCre expression by replacing the entire coding region of the prepro-Qrfp sequence in exon 2 of the Qrfp gene with iCre and the pgk-Neo cassette. Chimeric mice were mated with C57BL / 6J females (Jackson Labs). The pgk-Neo cassette was removed by mating with FLP66 mice that had been backcrossed to C57BL / 6J mice at least 10 times. First, F1 hybrids were generated from heterozygotes mated with heterozygotes. These mice were backcrossed to C57BL / 6J mice at least 8 times.

[0078] Rosa26 dreaddm3 and Rosa26 dreaddm4 Mice were generated by homologous recombination in C57BL / 6N embryonic stem cells, and then the same procedure as described above for Qrfp-iCre mice was performed.

[0079] (2) Virus AAV is as mentioned earlier. 33 The virus was prepared using triple transfection and a helper-free method. The final purified virus was stored at -80°C. The titer of the recombinant AAV vector was measured by quantitative PCR. AAV 10 - EF1α-DIO-TVA-mCherry, 4 x10 13 AAV 10 -CAG-DIO-RG, 1x10 13 AAV 10 - EF1α-DIO-hM3Dq-mCherry, 1.64 x10 12 AAV 10 - EF1α-DIO-mCherry, 1.44 x10 12 AAV 10 -EF1α-DIO-SSFO-EYFP, 1.35 x10 12 AAV 2 / 9 -hsyn-DIO-TeTxLC-GFP, 6.24 x10 14AAV 2 / 9 -hsyn-DIO-GFP, 4 x10 12 Genome copies / ml. Recombinant rabies vectors were prepared using previously reported methods. 22,34 The titer of SADΔG-GFP(EnvA) is 4.2 × 10⁻⁶. 8 The infection unit was / ml.

[0080] (3)Surgery For AAV vector injection, male Qrfp-iCre heterozygous mice (8-12 weeks old) were anesthetized with isoflurane and placed in a stereotactic frame (David Kopf Instruments).

[0081] For chemogenetic manipulation, AAV was administered to Qrfp-iCre mice. 10 -EF1α-DIO-hM3Dq-mCherry was injected into the hypothalamus (for MB injection, anterior-posterior direction (AP), -0.46 mm; medial-lateral direction (ML), ±0.25 mm; dorsal-ventral direction (DV), -5.75 mm; 0.50 μl at each site; LH injection; AP, -1.00 mm; ML, ±1.00 mm; DV, -5.00 mm; 0.30 μl at each site) at a rate of 0.1 μm / min using a Hamilton injection needle. The needle was left in place for 10 minutes after injection.

[0082] For optogenetic manipulation, AAV10-EF1α-DIO-SSFO-EYFP was unilaterally injected into AVPe (AP, 0.38 mm; ML, 0.25 mm; DV, -5.50 mm from bregma). Subsequently, optical fibers were implanted bilaterally above AVPe (AP: 0.38 mm, ML: ±0.25 mm, DV: -5.20 mm), bilaterally on DMH (AP: -1.70 mm, ML: ±0.25 mm, DV: -4.75 mm), or unilaterally on RPa (AP: -6.00 mm, ML: 0.00 mm, DV: -5.50 mm) (Figure 2j). After a recovery period of at least two weeks in individual cages following injection, mice were subjected to infrared thermal imaging experiments. Behavioral data were included only if these viruses specifically targeted Q neurons and the optical fiber implants were precisely positioned.

[0083] (4) Recording of Biological Signals For thermographic analysis, mice were placed in an experimental cage (25 × 15 × 10 cm) and monitored using an infrared thermal imaging camera (InfReC R500EX; NIPPON AVIONICS) positioned 30 cm above the cage floor. To clearly detect the surface temperature, the dorsal hair was removed with a hair clipper one day before the start of the experiment. Thermograms of DREADD and optogenetic experiments were acquired at 0.5 Hz and 1 Hz respectively, and analyzed with InfReC Analyzer NS9500 Professional software (NIPPON AVIONICS). The maximum temperature of one frame was used as the animal's T S (Figure 1d).

[0084] To record core body temperature, oxygen consumption, EEG, ECG, and respiratory patterns, each animal was housed in a temperature-controlled chamber (HC-100, Shin Factory, or LP-400P-AR, Nihon Ikaki Seisakusho Co., Ltd.). T B (intraperitoneal temperature) was continuously recorded by implanting a telemetry temperature sensor (TA11TA-F10, DSI) into the abdominal cavity of the animal under general inhalation anesthesia at least 7 days before recording. The animal's VO2 and carbon dioxide excretion rate (VCO2) were continuously recorded with a respiratory gas analyzer (ARCO-2000 mass spectrometer, ARCO System). The respiratory quotient was calculated from the VCO2 / VO2 ratio.

[0085] EEG and ECG were recorded using an implantable telemetry transmitter (F20-EET or HD-X02, DSI). For EEG recording, two stainless steel screws (1 mm diameter) were soldered to the telemetry transmitter wire and inserted into the cortical skull (AP, 1.00 mm; right, 1.50 mm from the bregma or lambda) under general anesthesia. The other two wires from the transmitter were placed on the surface of the thoracic cavity to record the ECG. Patients were allowed at least 10 days to recover from the surgery. The EEG / ECG data acquisition system consisted of a transmitter, an analog-to-digital converter, and a recording computer with the Ponemah Physiology Platform software (version 6.30, DSI). The sampling rate was 500 Hz for both EEG and ECG, and the data was converted to ASCII format for review. Heart rate was assessed by visual inspection of the waveform.

[0086] Respiratory flow is a non-invasive respiratory flow recording system. 35 The data was recorded as follows: Specifically, mice were placed in a metabolic chamber (TMC-1213-PMMA, Minamidrika Shokai) with an airflow of at least 0.3 L / min. The chamber was connected to a pressure sensor (PMD-8203-3G, Biotex) to detect the pressure difference between the outside and inside of the chamber. When an animal is breathing, the pressure difference from outside to inside increases during inhalation and decreases during exhalation. 35 The analog signal output from the sensor was digitized at 250Hz using an AD converter (NI-9205, National Instruments) and saved to a computer using data logging software developed by Biotex.

[0087] (5) FIT guidance The experiment to induce diurnal torpor was designed to record the animals' metabolism for at least three days. The animals were introduced into the chamber the day before recording began (day 0). Food and water were freely available. Awas set as shown on day 0 and kept constant throughout the experiment. The telemetry temperature sensor implanted in the mouse was powered on before being placed into the chamber. The standard experimental design was as follows. On day 2, at ZT-0, food was removed to induce daily torpor. 24 hours later, on day 3, at ZT-0, food was returned to each animal.

[0088] (6) Recording of metabolism during drug administration Clozapine N-oxide (CNO, a DREADD agonist, Abcam, ab141704) was dissolved in physiological saline at a dose of 100 μg / mL and frozen at -20°C. The CNO solution was thawed on site, and the solution was intraperitoneally administered to mice at a dose of 1 mg / kg. Adenosine A1 CHA (N 6 -cyclohexyladenosine, an adenosine receptor agonist, Sigma-Aldrich, C9901) was dissolved in physiological saline at a concentration of 250 μg / mL and intraperitoneally administered to mice at a dose of 2.5 mg / kg.

[0089] (7) Recording of metabolism during general anesthesia The aforementioned T B , VO2 and video recording (see "Recording of biological signals"), the inlet of the metabolic chamber was directly connected to the outlet of an inhalation anesthetic machine (NARCOBIT-E, Natsume Seisakusho Co., Ltd.). 1% isoflurane was administered at T A =28°C for 30 minutes, followed by T A =12°C for 90 minutes. After the experiment, the animals were warmed on a hot plate, and recovery was confirmed.

[0090] (8) Immunohistochemical staining Mice were deeply anesthetized with isoflurane, perfused transcardially with 10% sucrose in water, followed by perfusion with ice-cold 4% paraformaldehyde (4% PFA) in 0.1M phosphate buffer pH 7.4, and the brain was removed. The brain was fixed overnight in 4% PFA at 4°C, incubated overnight in 0.1M phosphate-buffered saline pH 7.4 (PBS) and 30% sucrose at 4°C, immersed in Tissue-Tek OCT compound (Sakura) in cryomold, and frozen at -80°C until sectioning. Using a cryostat (CM1860, Leica), the brain was coronally sliced ​​into four equal series at 50 μm intervals, collected in a 6-well plate filled with ice-cold PBS, and washed three times with PBS at room temperature (RT). Unless otherwise noted, the following incubation steps were performed with gentle shaking on an orbital shaker. Brain sections were incubated in 1% Triton X-100 in PBS at room temperature for 1 hour. Sections were blocked in 10% Blocking One (NACALAI TESQUE) in 0.3% Triton X-100-treated PBS (blocking solution) at room temperature for 1 hour without shaking. Sections were incubated overnight at 4°C in primary antibodies diluted with blocking solutions (dilutions and types of antibodies are shown below), then washed three times, incubated overnight at 4°C with secondary antibodies, washed in PBS, mounted, and covered with coverslips using HardSet Antifade Mounting Medium (VECTASHIELD) containing DAPI.

[0091] The primary antibodies used in this study were rabbit anti-cFos (1:4000, ABE457, Millipore), goat anti-mCherry (1:15000, AB0040-200, SICGEN), rat anti-GFP (1:5000, 04404-84, NACALAI TESQUE), mouse anti-TH (1:1000, sc-25269, Santa Cruz Biotechnology), mouse anti-orexin A (1:200, sc-80263, Santa Cruz Biotechnology), and rabbit anti-MCH (1:2000, M8440, SIGMA). The secondary antibodies are as follows: Alexa Fluor 488 donkey anti-rat, 488 donkey anti-rabbit, 594 donkey anti-rabbit, 594 donkey anti-goat, 647 donkey anti-mouse, and 647 donkey anti-rabbit (1:1000, Invitrogen). For Nissl staining, sections were counterstained with NeuroTrace 435 / 455 Blue Flue Fluorescent Nissl Stain (1:500, N-21479, Invitrogen) during the secondary antibody step and covered with a coverslip using FluorSave Reagent (Millipore). Brain regions were identified by Paxinos and Franklin. 36 This was determined using mouse brain maps.

[0092] (9) in situ hybridization Fluorescence in situ hybridization was performed using the RNAscope Fluorescent Multiplex Kit (Advanced Cell Diagnostics) with probes designed for RNAscope Fluorescent Multiplex in situ hybridization (ACDBio RNAscope Probe-Mm-Qrfp#4643411, mCherry#43201, Mm-Slc32a1#319191, Mm-Slc17a6#319171). The brain was dissected and immediately frozen in 2-methylbutane on dry ice and stored in cryogenic embedding medium at -80°C. Prior to dissection, the brain was cooled to -16°C for 1 hour in a cryostat. The brain was dissected into 20 μm coronal sections using a cryostat (Leica CM1860UV) and mounted on Superfrost Plus Microscope slides (Fisherbrand). The sample preparation and RNAscope Fluorescent Multiplex Assay were performed precisely in accordance with the RNAscope Assay Guide (document numbers 320513 and 320293, respectively).

[0093] (10) Retrograde tracking of Q neurons Male Qrfp-iCre mice (10-12 weeks old) were injected with the following virus: AAV 10 -DIO-TVA-mCherry and AAV 10 -DIO-RG was delivered to express TVA-mCherry and RG in Q neurons in the MB region (see above for procedure and coordinates). Two weeks later, SADΔG-GFP (EnvA) was injected into the same site. Starter neurons and input (single GFP-positive) neurons were detected in whole brain sections using a Leica TCS SP8 laser confocal microscope and a Zeiss Axio Zoom.V16, respectively.

[0094] (11) Blood chemistry tests Blood was collected from anesthetized mice by left ventricular paracentesis using a 25-gauge needle. The collected blood was not stored on ice for more than 2 hours. The samples were centrifuged at 2,000G for 10 minutes at 4°C, the supernatant was collected, and frozen at -30°C. The frozen serum samples were sent to FUJIFILM Wako Pure Chemical Corporation, where the concentrations of Na (mEq / L), K (mEq / L), Cl (mEq / L), AST (IU / L), ALT (IU / L), LDH (IU / L), CK (IU / L), GLU (mg / dL), and total ketone bodies (μmol / L) were measured.

[0095] (12) Electrophysiological analysis Mice were decapitated under deep anesthesia with isoflurane (Pfizer). The brains were extracted and cooled in an ice-cold cutting solution containing the following (mM): 125 mM choline chloride, 25 mM NaHCO3, 10 mM D(+)-glucose, 7 mM MgCl2, 2.5 mM KCl, 1.25 mM NaH2PO4, and 0.5 mM CaCl2 bubbled with O2 (95%) and CO2 (5%). Horizontal brain slices (250 μm thick) containing the hypothalamus were prepared using a vibratome (VT1200S, Leica) and maintained at room temperature for 1 hour in artificial CSF (ACSF) containing the following (mM): 125 mM NaCl, 26 mM NaHCO3, 10 mM D(+)-glucose, 2.5 mM KCl, 2 mM CaCl2, and 1 mM MgSO4 bubbled with O2 (95%) and CO2 (5%). Electrodes (5-8 MΩ) were packed with an internal solution containing the following (mM): 125 mM K-gluconate, 10 mM HEPES, 10 mM phosphocreatine, 0.05 mM tolbutamide, 4 mM NaCl, 4 mM ATP, 2 mM MgCl2, 0.4 mM GTP, and 0.2 mM EGTA (pH 7.3, adjusted with KOH). The firing of hM3Dq-mCherry-expressing neurons was recorded in current-clamp mode at a temperature of 30°C. The effect of CNO (1 μM) was investigated by applying it in a bath. Membrane voltage and data acquisition were controlled using a combination of a MultiClamp 700B amplifier, a Digidata 1440A A / D converter, and Clampex 10.3 software (Molecular Devices).

[0096] (13) 3D imaging of a transparent mouse brain As already mentioned, the transparent mouse brain 37The scales were prepared using the ScaleS method. Scale solutions were prepared using urea crystals (Wako Pure Chemical Industries, 217-00615), D(-)-sorbitol (Wako Pure Chemical Industries, 199-14731), methyl-β-cyclodextrin (Tokyo Chemical Industries, M1356), γ-cyclodextrin (Wako Pure Chemical Industries, 037-10643), N-acetyl-L-hydroxyproline (Skin Essential Actives, Taiwan), dimethyl sulfoxide (DMSO) (Wako Pure Chemical Industries, 043-07216), glycerol (Sigma, G9012), and Triton X-100 (Nacalai Tesque, 35501-15). Brains of Qrfp-iCre mice injected with AAV-DIO-GFP were fixed and cleared using ScaleS. The images were obtained using a laser confocal microscope (Olympus, XLSLPN25XGMP (NA 1.00, WD: 8mm) (RI: 1.41~1.52)).

[0097] (14) Statistical analysis In this study, Bayesian statistics was applied to evaluate the inventors' hypothesis and experimental results. The inventors designed a statistical model with parameters representing the structure of the hypothesis and fitted the model to the experimental results. Bayesian inference estimates the posterior probability distribution of model parameters from the likelihood distribution and prior probability distribution of the parameters. The posterior distribution provides information about how the model can explain the hypothesis from the experimental results. Bayesian models can explicitly include all types of uncertainty and therefore can handle data with noise in observations, or it can make full use of information from a small number of samples that may have a wide range of uncertainty. Furthermore, it can handle multiple layers of multiple groups with different numbers of samples using a hierarchical model. All these advantages of Bayesian inference make it suitable for addressing problems commonly encountered in animal experiments. Model fitting was performed in R version 3.52. 39 RStan library 38This was performed using Hamiltonian Monte Carlo with an adaptive variant, a non-U-turn sampler, to run on Stan version 2.18.0. Examination of the trace plots was also performed.

number

[0098] Convergence was evaluated by estimating the effective sample size. The prior probability density function of the model is defined by weak informability and conservatism, and is specified in the following section. The basic principles and techniques of statistical model design are described in the book Statistical Rethinking. 40 This is based on [the following]. The source code for both the model and data used in the analysis is available at https: / / briefcase.riken.jp / public / JjtgwAnqQ81AgyI. (For evaluation purposes, it will be made public, protected by the password "qih").

[0099] Body weight of Qrfp-iCre mice was modeled using a state-space hierarchy model (code folder QRFP_KO_BW) for specified ages and strains. Each group of Qrfp-iCre mice—wild-type (n = 9), heterozygous (n = 9), and homozygous (n = 10)—was housed in separate cages without individual identification. An unobservable baseline of body weight was defined using the time variable B. t,s We define the trend η as follows, where t is time, and the lineage indicators (1, 2, and 3 for wild-type, heterozygous, and homozygous Qrfp-iCre mice, respectively) are used to define the trend η. t , s And expressed in terms of total time point T, the observed state Y t,i This can be described by modeling the observation error using a log-normal distribution as follows.

[0100]

number

[0101] A uniform prior probability density function was applied to all parameters except σ1 and σ2, which were derived from a standard half-normal distribution.

[0102] The spike frequency of Qrfp-positive neurons in brain slices was modeled by parameterizing the difference in spike frequency when neurons were activated by CNO (code folder Patch_M3_CNO). The total number of slices is K, and the observed spike frequencies of the control and CNO administration recordings for the i-th slice are respectively B i and C i , B i is modeled by β with observation error BASE , and C i is modeled by the sum of β with observation error BASE and β CNO . Since the spiking frequency is a positive real number, the error can be modeled by a log-normal distribution, thus B i and C i can be described as follows.

[0103] [Formula]

[0104] All σ were sampled from a standard half-normal distribution.

[0105] T of the photostimulated animals S was modeled with a hierarchical multilayer model (Figure 21, code folder SSFO_Opto). Four groups of animals were included in this experiment. T S was recorded at 1 Hz, the median value was saved every 10 seconds for further analysis. T recorded 115 to 125 minutes after the first photostimulation S was all included in the analysis. Where K is the total number of animals, and Y is T during the time of interest for mouse j belonging to i S , Y is modeled with observation noise following a Cauchy distribution with scale parameter σ ERROR , accompanied by the global mean parameter β and the group parameter β GROUP, and individual mouse parameters β MOUSE It can be expressed as the sum of [the specified values].

[0106]

number

[0107] All σ were sampled from a standard semi-normal distribution. S The difference between groups is σ MOUSE β and β have normal distribution noise with standard deviation GROUP From the posterior distribution, which is the sum of the mean T values ​​for each group, S We estimated and compared them.

[0108] To evaluate the thermoregulatory system under QIH and normal conditions, the heat loss and heat production of animals were described using a hierarchical multilayer model (Figure 3c-k, code folder QIH_GTRH). Three parameters G, T were used under two metabolic conditions, namely normal and QIH. R and H, various T A This was estimated from the metabolically stable state of animals in the study. The detailed method was described above. 3 In short, the controllable parameter T A and the observable parameter T B A linear model consisting of and VO2 is used, with T as a predictor having normally distributed noise. A Using T B The experimental results for both and VO2 were fitted. Next, the posterior distributions of the slope and intercept coefficients of each model were used to determine G, T R , and H were estimated. In this analysis, the prior probability density function of the standard deviation of the noise is a standard semi-normal distribution, and the other parameters use a uniform distribution resulting in negative values ​​for T B We used the positive region of a uniform distribution, excluding the intercept coefficient.

[0109] Circadian metabolic changes in Q-TeTxLC mice were analyzed by modeling metabolism by clustering recorded values ​​into L and D phases (code folder TeTxLC_LD). In particular, Y represents the observed T in group i of phase j.B In this case, Y can be expressed as the sum of the differences between basal metabolism (L-phase metabolism) and D-phase metabolism, and the observational noise of a normally distributed system is as follows:

[0110]

number

[0111] All σ values ​​were sampled from a standard semi-normal distribution. In the VO2 modeling, since VO2 is assumed to be only positive real numbers, the basic model structure is T, except that the observation error is modeled as a log-normal distribution. B It was identical to the modeling. Metabolism during FIT in Q-TeTxLC mice was modeled using a hierarchical multilayer model (Figure 4d, code folder TeTxLC_FIT). The minimum value Y of a certain group i in section j is equal to group β. 0[i] Average metabolism and difference parameter β 1[i,j] It can be expressed as the sum of [the specified values].

[0112]

number

[0113] To model metabolic variance, mouse identity was included as a predictor of the observed value Y. In this way, Y for a given group in a section was modeled as a normal distribution, and this normal distribution has a mouse-dependent mean α. MOUSE and group and section-dependent parameter β GROUP,SECTION Using σ as the mean, GROUP,SECTION This was used as the standard deviation.

[0114]

number

[0115] All σ in equations (22)-(24) and (28)-(30) were sampled from a standard semi-normal distribution. These models are TB This model was used for modeling VO2 and RQ. Even Y in these models can theoretically accept negative real numbers, and the latter converged well, so this model was also applied to VO2 and RQ.

[0116] [Experiment and Results]

[0117] Example 1: Induction of metabolic depression by a chemically defined population of hypothalamic neurons Pyroglutaminated RF amide peptide (QRFP), a hypothalamic neuropeptide, was originally discovered through a bioinformatics approach aimed at discovering novel RF amide peptides. 9,10 The Qrfp peptide was also identified and purified from rat brain as an endogenous ligand for the orphan G protein-binding receptor hGPR103. 11 Prepro-Qrfp mRNA is localized only in the hypothalamus, specifically in the periventricular nucleus (Pe), lateral hypothalamic area (LHA), and gray ridge (TC). 11 It is distributed in [location]. Qrfp has been thought to be involved in food intake, sympathetic nerve regulation, and anxiety. 11,12 The inventors created mice (Qrfp-iCre mice) in which codon-modified Cre recombinase (iCre) was knocked into the Qrfp gene. The inventors also created mice (Qrfp-iCre;Rosa26) in which hM3Dq-mCherry is expressed only in iCre-expressing neurons. dreaddm3 To obtain a mouse, CAG-hM3Dq-mCherry was inserted into the Rosa26 locus with an upstream floxed transcriptional stop element. ddreadm3 It was crossbred with a mouse. Qrfp‐iCre;Rosa26 dreaddm3 During excitatory chemogenetic experiments using mice, these mice showed a marked decrease in motor activity, ultimately entering a severe and persistent state of immobility that began approximately 30 minutes after intraperitoneal (IP) injection of clozapine-N-oxide (CNO). Noting that the posture of these mice was similar to that observed during diurnal tropor, we concluded that activation of iCre-positive cells in Qrfp-iCre mice induces a diurnal tropor-like state, resulting in immobility and low T BWe initially hypothesized that this would be a characteristic of the condition (as will be discussed later, it has become clear that the hypothermia induced here is not diurnal dormancy but a hibernation-like state). To evaluate this hypothesis, we used a thermographic camera to measure surface body temperature (T S ) measure Qrfp-iCre;Rosa26 dreaddm3 We found that CNO-induced immobility in mice is accompanied by marked and persistent hypothermia (Figure 1b). S The decrease began approximately 5 minutes after CNO administration and lasted for nearly 12 hours. Subsequently, the mice spontaneously recovered from hypothermia without external reheating.

[0118] In contrast, Qrfp-iCre;Rosa26 dreaddm4 Inhibitory DREADD manipulation via hM4Di activation in mouse iCre-positive neurons is T S It showed no effect whatsoever (Figure 1b). Importantly, Qrfp-iCre;Rosa26 dreadm3 hM3Dq-mediated activation of iCre-positive neurons in mice induced severe hypothermia even in homozygous Qrfp-iCre mice in which the prepro-Qrfp sequence was completely replaced with iCre in both alleles (Figure 1b). This suggests that the Qrfp peptide itself is not essential for inducing hypothermia. Rather, the degree of hypothermia was more pronounced in Qrfp knockout (Qrfp-iCre homozygous) mice, suggesting that endogenous Qrfp itself may counteract hypothermia. This contradicts the inventors' previous observation that Qrfp increases sympathetic outflow and raises heart rate and blood pressure upon central administration. 11 This matches.

[0119] Therefore, we identified Qrfp as a chemical marker for hypothermia-inducing neurons. Next, since iCre-positive neurons are observed only in the hypothalamus, but are distributed in several discrete hypothalamic regions in Qrfp-iCre mice, we attempted to identify the hypothalamic region that induces hypothermia. Two different stereotactic coordinates; medial basal (MB) injection or lateral (LH) injection (see Methods) and a flip-excision (FLEX) switch.13 By injecting a Cre-activated AAV vector containing into the hypothalamus of Qrfp-iCre mice, iCre-positive neurons in the lateral and medial regions of the hypothalamus were manipulated separately. MB injection of Cre-dependent AAV vector enabled the expression of specific genes in iCre-positive neurons in the medial region of the hypothalamus, namely the anterior ventral periventricular nucleus (AVPe), medial preoptic area (MPA), and Pe, but not in LHA (Figure 1c). Multicolor fluorescence in situ hybridization analysis confirmed that most mCherry-positive cells in these regions expressed Qrfp mRNA. 10 -EF1a-DIO-hM3Dq-mCherry was injected via MB to express hM3Dq in this region, and electrophysiological studies were performed using hypothalamic slices prepared from these mice to confirm that CNO bath application strongly excited mCherry-positive neurons. IP injection of CNO into these mice resulted in Qrfp-iCre;Rosa26 dreaddm3 It was found to cause deeper and longer-lasting hypothermia than the severe immobility observed in mice (Figure 1b, Figure 1d). Very low T B The condition (below 30°C) lasted for more than 48 hours (Figure 1d). Immunostaining with anti-Fos and anti-mCherry antibodies revealed numerous mCherry and Fos-positive neurons in AVPe, MPA, and Pe, and in vivo excitation of these neurons by CNO was confirmed (Figure 1e).

[0120] From these observations, we concluded that iCre-positive neurons in AVPe / MPA and Pe in Qrfp-iCre mice (these neurons will be later referred to as resting-induced neurons or Q neurons) are primarily responsible for induced hypothermia. In the following experiments, unless otherwise specified, the inventors used AAV for the induction of hypothermia. 10 We primarily used Qrfp-iCre mice with MB injections, specifically the -EF1a-DIO-hM3Dq-mCherry (referred to as Q-hM3D mice).

[0121] To further analyze induced hypothermia, a remote temperature sensor was implanted in the peritoneal cavity of Q-hM3D mice, and metabolism was continuously analyzed by respiratory gas analysis (Figure 1f). This study found that CNO-induced hypothermia in Q-hM3D mice was accompanied by a significant decrease in the rate of O2 consumption (VO2: oxygen consumption) (Figure 1g), and that T after CNO administration S Together with T B We confirmed that both decreased simultaneously. In contrast, AAV 10 Excitatory DREADD manipulation of iCre-positive neurons in the lateral hypothalamic region (LHA and TC) of Qrfp-iCre mice by LH injection of -EF1a-DIO-hM3Dq-mCherry did not induce hypothermia (Figure 1g).

[0122] During Q neuron-induced hypothermia / hypometabolism (QIH), heart rate decreased significantly (758 beats / min and 215 beats / min 2 hours before and 2 hours after CNO injection, respectively) (mean of n=3). Respiratory rate decreased from 333 breaths / min to undetectable levels (tidal volume was below the detection limit). At these times, VO2 decreased from 3.60 to 1.17 ml / g / hr. During QIH, mice exhibited very low-amplitude electroencephalogram (EEG), which was distinctly different from that observed in non-rapid eye-movement sleep characterized by high-amplitude slow waves. Serum chemistry data suggested decreased blood glucose levels during QIH, likely due to reduced gluconeogenesis resulting from decreased sympathetic tone. These observations further suggest that many bodily functions are impaired during QIH. B This suggests that it decreases robustly along with a decrease in VO2.

[0123] The effects mediated by DREADD typically last only a few hours after CNO injection, but DREADD-induced QIH in Q-hM3D mice persisted for a very long time. Surprisingly, T A =20℃, T below 30℃ BQIH persisted for over 48 hours after a single dose of CNO (1 mg / kg), and it took approximately one week for VO2 to fully recover to normal (Figure 1h). After recovery from QIH, the mice were healthy and appeared to behave normally. QIH was reproducible in the same mice after repeated CNO injections, demonstrating the reversibility of this procedure (Figure 1h).

[0124] Example 2: Q neurons act on the dorsomedial hypothalamus to induce QIH. To elucidate the mechanism that induces QIH, we analyzed the axonal projections of Q neurons. AAV was applied to Qrfp-iCre mice. 10 After injecting EF1a-DIO-GFP to specifically express GFP in Q neurons (Figure 2a, b), GFP-positive fibers were observed in the MPA, VOLT, paraventricular nucleus (PVN), supraoptic nucleus (SON), dorsomedial hypothalamus (DMH), LHA, tuberomammillary nucleus (TMN), medial mammillary nucleus (MM), periaqueductal gray matter (PAG), lateral parabrachial nucleus (LPB), locus coeruleus (LLC), rostral ventrolateral area of ​​the medulla oblongata (RVLM), and pallid raphe nucleus (RPa) (regions involved in thermoregulation and sympathetic nerve control) (Figure 2c). 14 The inventors found that DMH received particularly abundant projections. Brain analysis using the ScaleS method further suggested the location of projections from Q neurons to DMH (Figure 2d). Next, we used triple-color in situ hybridization of Q neurons to determine whether they were inhibitory or excitatory. After confirming that CNO injection effectively induced QIH in Q-hM3D mice, these mice were subjected to in situ hybridization histochemical analysis. We used transcripts encoding mCherry, an excitatory and inhibitory marker, and probes encoding vesicular glutamate transporter 2 (Vglut2) and vesicular GABA transporter (Vgat). We found that approximately two-thirds of the Q neurons were Vgat-positive and approximately two-fifths were Vglut2-positive (Figure 2e-i).

[0125] Within the region containing abundant projections from Q neurons (Figure 2c), we focused on DMH because thermogenic neurons had previously been identified in DMH. 15 To elucidate the function of Q neuron axonal projections to the DMH, an optogenetic approach was used. AAV was applied to Qrfp-iCre mice (Q-SSFO mice). 10 -DIO-SSFO-eYFP injection stabilizes step function opsin (SSFO) in Q neurons. 16 We expressed (Figure 2j). We confirmed that SSFO is expressed in AVPe, MPA, and Pe. S To confirm the effect of optogenetic excitation on T, we first implanted an optical fiber into AVPe / MPA, where many Q neuron cell bodies are found (Figure 2j), and applied an optical pulse (1-second wide optical pulse) to photogenetic excitation of SSFO-positive cell bodies in mice. Under these conditions, optogenetic excitation of Q neurons rapidly induced severe hypothermia, which lasted for about 20 minutes (Figure 2k). When Q neurons were excited four times at 30-minute intervals, T A (22℃) S Marked hypothermia accompanied by [unclear] was observed. Many Fos-positive neurons were identified in SSFO-eYFP-positive cells of AVPe / MPA after excitation (Figure 2j). Optogenetically induced QIH was significantly shorter in duration than QIH induced by hM3Dq-mediated pharmacogenetic excitation of Q neurons (Figure 2k), suggesting that Gq-mediated metabolic regulatory signaling in Q neurons, which leads to changes in gene expression profiles, may play a role in creating the long-term persistence of QIH.

[0126] Next, optical fibers were implanted in the DMH on both sides of the Q-SSFO mice, and light stimulation was applied to the axonal fibers. This procedure effectively T S This reduced the activity, but only slightly less than that induced by AVPe / MPA cell body stimulation (Figure 2k,l). In contrast, RPa is known to contain sympathetic premotor neurons for thermogenesis mediated by brown adipose tissue regulation. 17Therefore, we also investigated the effects of light stimulation on Q neuron fibers in RPa. S A subtle effect of photogenetic excitation of Q neuron fibers on RPa was also observed (Figure 2k,l). From these results, we hypothesized that Q neurons primarily act on DMH, and to a smaller extent act on RPa, inducing QIH.

[0127] Example 3: The theoretical set temperature decreases during QIH. An increase in mouse tail temperature was observed immediately after QIH induction, and since it was induced by optogenetic or pharmacogenetic excitation of Q neurons, T B It was suggested that peripheral blood vessels dilate and release heat during the decrease in T (Figure 1d, Figure 2k). B Peripheral vasodilation without an increase in the theoretical body temperature setpoint (T) is observed in hibernating animals during hibernation. R This suggests that the thermoregulatory system is reset to a lower value than normal. To evaluate this, we performed a characteristic analysis of the thermoregulatory system of mice during QIH. Under conditions where the animal has no external work and its metabolism is stable, multiple ambient temperatures (T A ) Below, T B and from VO2, the thermal conductance (G), H and T R It is possible to estimate 3 Q-hM3D mice were prepared, and various T A QIH under (8, 12, 16, 20, 24, 28 and 32°C) B And VO2 was recorded (Figure 3a). 11-hour mean T after IP injection of physiological saline or CNO. B And VO2 was compared. During QIH, the animals compared all T with the corresponding controls. A low T B This shows VO2 (Figure 3b). When the heat production system is functioning properly, i.e., T R is T B At higher temperatures, the thermoregulatory system increases VO2. R When attempting to reach (Figure 3c), T A As T increases B T increases, and VO2 decreases. B VO2 and each have different TA The minimum value is shown at T A Therefore, only the coordinated heat generation characteristics in the range of 16-24°C were shown (Figure 3b). Thus, T in QIH A Further analysis was performed using metabolic data at 16, 20, and 24°C. First, T B -T A G was estimated from the relationship between G and VO2 (Figure 3d). The 89% peak posterior density interval (HPDI) of G was [0.212,0.221] ml / g / hr / ℃ and [0.182,0.220] ml / g / hr / ℃ under normal and QIH conditions, respectively (Figure 3e; hereafter, 89% HPDI is shown in two numbers in square brackets). Quantitatively, the posterior distribution of the difference between the two Gs (ΔG) was [-0.0040,0.0348] ml / g / hr / ℃ (Figure 3f), which includes 0, suggesting that G under normal conditions and G under QIH conditions cannot be distinguished. This differed from diurnal dormancy (torpor), which shows a lower G than normal conditions. 3 Secondly, T B And VO2 to H and T R The following was estimated (Figure 3g). H was [3.43, 8.72] ml / g / hr / ℃ in the normal state and [0.181, 0.369] ml / g / hr / ℃ in QIH (Figure 3h), representing a 95.3% decrease in each median. The posterior distribution of the difference (ΔH) was [3.17, 8.48] ml / g / hr / ℃ (Figure 3i), which was positive, suggesting that there is an 89% or greater probability that these conditions are different. This decrease in H was similar to the decrease in H during fasting-induced diurnal rest (FIT). 3 In particular, T R The estimated temperatures were [36.04, 36.60]°C under normal conditions and [26.83, 29.13]°C under QIH (Figure 3j). R The difference is 8.41°C for each median, and the posterior distribution of the difference (ΔT R ) is [7.18,9.57]℃, and T in QIH R This clearly shows a decrease (Figure 3k). Very small theoretical set temperature shift in FIT. 3 Considering this, this observation highlights the similarities between QIH and hibernation, as well as the differences between QIH and diurnal torpor.

[0128] A prominent characteristic of hibernation is T in QIH R To provide further evidence of the decrease, during QIH, T A When QIH changed dynamically, we observed the relationship between posture and metabolism within individual mice (n=4, representative data from one mouse in Figure 3l, and data from the other three). The highly stable and prolonged low-metabolic state of QIH, similar to that in hibernating animals, made it possible to investigate this in mice. Q-hM3D mice were T A The temperature was set to 28°C to induce FIT (Figure 3l A and B). After 24 hours of recovery, QIH was induced by CNO administration (Figure 3l C, D, E, and F). Interestingly, T A At 28°C, the mice exhibited an elongated posture during QIH, a posture typically seen in animals exposed to high-temperature environments (Figure 3l, D). This indicates that T A This was clearly different from the typical sitting posture observed during FIT at 28°C (Figure 3l, B). This behavioral observation further indicated T R This indicates that QIH is lower than FIT and normal conditions. Furthermore, T A When the temperature was lowered to 12°C, the animal returned to a sitting position similar to diurnal dormancy (torpor) (Figure 3l, E), and tremors began. These results indicate that during QIH, T R While physical function and behavior decline, T A I strongly support the idea that it will still be adjusted to adapt to the changes.

[0129] Animals have a low metabolic rate during hibernation, A It is well known that its metabolism is actively regulated in response to this. Similarly, in QIH, T below 16℃ A Animals exposed to the 20°C or 28°C T A Compared to animals exposed to T, it showed a considerably higher VO2 (Figure 3b). In fact, this is because T A Previous reports on hibernating animals showing increased metabolism when certain levels were lowered. 18 This is similar to the characteristic of active metabolic deficiency in QIH. This characteristic of active metabolic deficiency in QIH was also observed in individual animals (Figure 3l). The behavioral and metabolic responses of mice in QIH were similar to the physical function of T BThis was completely different from what was observed in a normal state where the body is trying to maintain a narrow range.

[0130] To compare metabolic function during QIH with that of anesthesia, the inventors used multiple T A Metabolic transitions during general anesthesia were recorded below. As expected, animals under anesthesia showed low T levels. A Exposure to the substance did not result in an increase in VO2 or a change in posture. We also investigated the metabolic state induced by systemic delivery of the adenosine A1AR agonist (6) N-cyclohexyladenosine (CHA), which is used to induce hypothermia. 19 IP injection of CHA (2.5 mg / kg) into wild-type mice effectively induced hypothermia / hypometabolism, but the mice exhibited either increased VO2 or behavioral changes (posture and tremors), resulting in low T. A (12℃) did not react. T at 20℃ B While T tended to be higher in CHA-induced hypothermia than in QIH, B And VO2 decreased further with CHA-induced hypothermia. On the other hand, T A When the temperature was set to 12°C, these parameters increased in QIH (Figure 3b). These observations indicate that QIH is completely different from general anesthesia or CHA-induced passive hypothermia, and that it is T B This study demonstrates that blocking the regulatory system induces hypothermia.

[0131] Example 4: Q neurons are involved in normal fasting-induced diurnal torpor. QIH resembles hibernation more than diurnal torpor, but since torpor is sometimes considered a milder form of hibernation, we investigated whether Q neurons are also involved in torpor. Furthermore, we explored the possibility that common or similar mechanisms may play a role in inducing both hibernation and torpor. 20 To investigate the role of Q neurons in circadian torpor, AAV was introduced into Qrfp-iCre mice (Q-TeTxLC mice). 2 / 9By injecting -hSyn-DIO-TeTxLC-eYFP, tetanus toxin light chain (TeTxLC) was specifically expressed in Q neurons, and we investigated whether blocking SNARE complex-mediated neurotransmission in Q neurons affected FIT (Figure 4b). AAV 2 / 9 -hSyn-DIO-TeTxLC-eYFP and AAV 10 Simultaneous injection of -EF1a-DIO-hM3Dq-mCherry is T B CN This completely eliminated the action of O, suggesting that blocking the SNARE complex eliminates the QIH-inducing ability of Q neurons. We found that the normal structure of FIT was disrupted in all Q-TeTxLC mice. No rapid metabolic oscillations were observed in these fasted mice (Figure 4c,d). This suggests that the function of Q neurons is impaired during FIT. B This suggests that it is necessary to induce a rapid decline in T. Interestingly, the T observed in these mice B The gradual decrease indicates the existence of a Q neuron-independent metabolic reduction mechanism during FIT. In addition, Q-TeTxLC mice showed higher T levels than control mice. B The circadian variation is small, B This suggests a major role for Q neurons in circadian regulation. In particular, homozygous Qrfp-iCre mice lacking the QRFP peptide showed normal FIT (Figure 4e). These observations suggest that Q neurons are essential components for inducing diurnal torpor and play a crucial role in the rapid shift in body temperature during torpor, but QRFP does not.

[0132] To elucidate the neuronal mechanisms that regulate Q neuron activity, we investigated recombinant pseudorabies virus vector (SADΔG(EnvA))-mediated labeling. 21 (Figure 4f) identifies the upstream neuronal population that directly synaps with Q neurons. Cre-activated AAV vector in Qrfp-iCre mice. 22After expressing TVA-mCherry and rabies glycoprotein (RG) in Q neurons using [a specific method], SADΔG-GFP (EnvA) was injected into the same site. Double-positive starter cells for TVA-mCherry and GFP were found in AVPe / MPA and Pe (Figure 4g). In the median preoptic nucleus (MnPO), PVN, and MPA, input neurons directly synapsing to Q neurons (GFP-positive but mCherry-negative) were identified (Figure 4h). Input neurons were also observed within and around AVPe / Pe, suggesting the presence of local interneurons that modulate Q neuron function and the possibility that Q neurons form microcircuits with interneurons within AVPe / MPA and Pe. These observations suggest that Q neurons receive relatively sparse direct input from the intrathalamic region. Since FIT is induced by fasting, Q neurons are expected to monitor the negative energy balance. Neurons in PVH have been shown to receive abundant input from ARC. 23 Therefore, input from PVH to Q neurons may play a role in transmitting information about nutritional status. PVH input may also transmit circadian information from the suprachiasmatic nucleus (SCN).

[0133] MPA is T B It is involved in the regulation of 24,25 Therefore, the interaction between Q neurons and MPA may play an important role in thermoregulation. VMPO also contains input neurons. In previous studies, thermosensitive neurons in the ventromedial preoptic area (VMPO) were identified as BDNF and PACAP bipositive neurons. Excitation of these cells also induced hypothermia. 26 This effect is much smaller than that induced by the excitation of Q neurons, but VMPO PACAP / BDNFFunctional interactions between neurons and Q neurons may exist. Furthermore, DREADD excitation of TRPM2-positive cells in the POA has been shown to induce hypothermia. Since TRPM2 is ubiquitously overexpressed in the POA, including regions containing AVPe / MPA and input neurons to Q neurons, TRPM2-induced hypothermia may be induced by direct and / or indirect activation of Q neurons. 27 .

[0134] Q neurons are localized along the third ventricle (3V), and their dendrites extend along the ependymia and periventricular regions of 3V (Figure 1c), potentially allowing them to sense humoral factors released by tanicates and ependymal cells, factors in the cerebrospinal fluid, or even capillaries.

[0135] [Consideration] Here, we demonstrate the existence of a novel hypothalamic neuron population in mice with specific chemical (=Qrfp expression) and histological (AVPe / MPA) characteristics, where excitation of this population induces an active metabolic depression very similar to hibernation. This state, QIH, shares two major properties with hibernation. One is T R One is a decrease in heart rate, and the other is a actively regulated low metabolism. During QIH, mice actively regulate their bodily functions in response to the external environment. Many other physiological parameters, such as decreased heart rate, weak respiration, and low-voltage electroencephalogram, suggest similarities between QIH and hibernation. 29 Fos expression analysis revealed that the thirteen-lined ground squirrel 30 Previous studies have shown that cells near 3V are activated during hibernation. This activation pattern is very similar to the region where Q neurons are localized, suggesting that hibernation neurons may also utilize Q neurons to induce hibernation.

[0136] The fact that mice were able to enter a hibernation-like state (=QIH) is quite surprising. While rodents, canis, and even distantly related mammals including primates are capable of hibernation, suggesting that the neural mechanisms of hibernation are conserved across a wide range of mammalian species, it is reasonable to assume that these systems are not normally recruited in non-hibernating animals. Since the Qrfp gene is also conserved in humans, it can be inferred that Q neurons may exhibit a low metabolic state of activity when excited. This study also confirmed that DMH is a major effector site for Q neurons. Future research identifying QIH-induced neurons in DMH will further elucidate the mechanisms of QIH. Q neurons may also act on other regions identified in this study. For example, SON has recently been reported to play an important role in general anesthesia and sleep. 32 .

[0137] Furthermore, we found that Q neurons are necessary for diurnal torpor induced by fasting in mice. However, we were unable to repeatedly detect an increase in Q neuron activity during fasting in mice using Fos staining or fiber optics (data not shown), suggesting that low levels of Q neuron activation may be sufficient to induce hypothermia during diurnal torpor.

[0138] The induced hibernation demonstrated in non-hibernating animals in this study represents a promising step forward in understanding the neuronal mechanisms of active metabolic depletion and provides a method for investigating how each tissue adopts a hibernation-like metabolic depletion state. Furthermore, with future developments in methods for selectively exciting Q neurons, QIH will provide a novel approach for developing methods that enable the clinical application of synthetic hibernation in humans, which would offer significant medical advantages, such as potentially reducing systemic tissue damage after heart attack or stroke, or being useful for organ transplant preservation.

[0139] When the above experiment was performed using rats instead of mice, it was possible to induce the QIH state. Done 43 Specifically, AAV10 -CaMKIIα-hM3Dq-mCherry was injected into the AVPe / MPA of rat brains. Addition of CNO activated the left and right AVPe / MPA, including Q neurons, inducing QIH in rats, accompanied by hypothermia and low metabolism. Rats with QIH exhibited decreased VO2 and an elongated posture. These characteristics were similar to those observed in QIH in mice. Selective activation of Q neurons also induced QIH in mammals that do not normally hibernate, such as rats.

[0140] Example 5: Treatment of acute diseases by hibernation This invention confirmed the effects of treating subjects with acute diseases by inducing hibernation. The acute diseases tested were sepsis and acute renal failure.

[0141] Sepsis was induced in the cecal ligation and paracentesis (CLP) model (see Figure 9A). The mice were anesthetized with isoflurane. A longitudinal midline incision was made in the skin with a scalpel, taking care not to enter the peritoneal cavity. After the initial incision, the incision was widened with small scissors. The cecum was removed and ligated in place. Before cecal perforation, the contents of the cecum were gently pushed toward the distal cecum, and during cecal perforation, trapped air and gas were gently aspirated. The cecum was perforated with a single penetrating puncture from the mesentery toward the anti-mesentery at a point midway between the ligation site and the tip of the cecum. After the needle was withdrawn, a small amount of excrement (droplets) was pushed out from both the mesenteric and anti-mesentery penetration holes to confirm patency. The feces from the cecum were repositioned into the abdominal cavity without spreading them to the abdominal wall wound margin. The peritoneum, fascia, and abdominal fascia were closed with simple running sutures. The surgery was performed under appropriate temperature control.

[0142] After CLP, hibernation (QIH) was induced in the mice. Hibernation was not induced in the negative control group. The survival rate of the mice was observed for 48 hours after CLP (see Figure 9A). The results are shown in Figure 9B. As shown in Figure 9B, the group in which hibernation was induced (QIH group) showed an extended survival period compared to the negative control group.

[0143] Figure 10 shows the time course of oxygen consumption in a sepsis model, a model in which hibernation was induced immediately after sepsis induction, and a typical case of QIH. In the sepsis model, oxygen consumption decreases over approximately 10 hours, whereas in hibernation induction, oxygen consumption decreases in about 1-2 hours. The hypometabolism induced in sepsis causes a decrease in oxygen consumption in about 1-2 hours, supporting the presence of QIH.

[0144] Acute renal failure was induced in a renal pedicle clamp model. As shown in Figure 11A, mice were subjected to general anesthesia by inhalation anesthesia. Skin incisions and abdominal wall myotomy were performed to expose the kidneys. The adipose tissue around the kidneys was dissected to expose the renal pedicle, which was then ligated. The wounds were then closed. After renal pedicle ligation, hibernation (QIH) was induced in the mice. Hibernation was not induced in the negative control group. The survival rate of the mice was observed for 48 hours from CLP. The results are shown in Figure 11B. As shown in Figure 11B, the group in which hibernation was induced showed an extended survival period compared to the negative control group.

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Claims

1. A device for monitoring hypometabolic states in subjects with disease and controlled hypometabolic states, A measuring unit that measures the oxygen concentration of exhaled and inhaled air from the subject, A calculation unit that calculates the oxygen consumption of the subject from the difference in oxygen concentration between the subject's exhaled and inhaled breath, A low-metabolic state monitoring unit monitors oxygen consumption over time, and if oxygen consumption decreases compared to before the low-metabolic state, it is estimated that the subject is in a low-metabolic state, and if oxygen consumption is zero, it is estimated that the subject has died or may have died. A deep body thermometer, a temperature storage unit that stores the temperature measured by the thermometer, a calculation unit that calculates a theoretical set temperature, and a calculation unit that calculates the heat generation feedback gain (H) A device including a device.

2. The apparatus according to claim 1, wherein the controlled low metabolic state is a hibernation-like state.

3. The apparatus according to claim 1 or 2, further comprising a determination unit that records fluctuations in oxygen consumption over a certain period for a subject in a stable low metabolic state, sets a predetermined range based on the fluctuations in oxygen consumption over that certain period, and determines whether the oxygen consumption is within or outside the predetermined range.

4. The system is further equipped with an output device that, if the oxygen consumption exceeds the upper limit of a specified range, outputs to the output device that the subject may be about to awaken from a controlled hypometabolic state; if the oxygen consumption falls below the lower limit of a specified range, outputs to the output device that the subject may be about to transition to an even lower metabolic state than the controlled hypometabolic state; and, if the oxygen consumption is within the specified range, outputs to the output device that the subject is in a controlled hypometabolic state. The apparatus according to any one of claims 1 to 3.

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