A method for positioning the vmhvl brain region of a cranial suture variant animal

Abstract

The application discloses a VMHvl brain area positioning method suitable for animals with cranial suture variation, and aims to solve the problems of large system error (ML offset 155.31+ / -25.57 um) and targeting failure of traditional fontanel positioning method of outbred animals such as Mongolian gerbils due to the ambiguity and bifurcation of the intersection of the sagittal suture and the coronal suture. The method takes the intersection of the sagittal suture and the herringbone suture as the posterior fontanel, and after the digital positioning instrument is zeroed, the positioning needle is moved from the estimated position of the anterior fontanel to the posterior fontanel along the Y axis; then the positioning needle is uniformly moved from the posterior fontanel to the anterior fontanel, and the visual intersection of the coronal suture is the anterior fontanel; then the anterior and posterior leveling is realized through the Z value difference between the anterior fontanel and the posterior fontanel being less than or equal to 0.03 mm, and the left and right leveling is realized through the Z value difference between the left point and the right point being less than or equal to 0.05 mm; finally, the anterior fontanel is taken as the origin, and the VMHvl is positioned according to ML+ / -0.75 mm, AP-1.7 mm and DV-7.5 mm. The application reduces the ML offset to 2.29+ / -0.96 um (P<0.001), the targeting qualification rate is 100%, and the coincidence rate is greater than or equal to 95%, so that the reliability of downstream experiments such as optogenetics and electrophysiology is significantly improved.

Description

Technical Field

[0001] This invention relates to the field of experimental methods in neuroscience research, and in particular to a method for locating the VMHvl brain region in animals with cranial suture variations. Background Technology

[0002] Stereotactic brain imaging is a core foundational technology in neuroscience for exploring the function of brain nuclei and analyzing the regulatory mechanisms of neural circuits. Its positioning accuracy directly determines the reliability and effectiveness of subsequent experiments such as brain region intervention, electrophysiological recording, and tracer labeling. The core support for this technology lies in the stability, identifiability, and spatial correspondence between anatomical landmarks on the skull surface and target nuclei in the brain.

[0003] Inbred experimental animals, due to their highly homogeneous genetic background and regular cranial suture development, with a clear and unique intersection of the coronal and sagittal sutures, allow for minimal systematic error control when using the anterior fontanelle for brain region localization. This method can meet the localization requirements of small functional nuclei such as the ventrolateral hypothalamus nucleus (VMHvl) and the median preoptic nucleus. However, in outbred rodents and some experimental animals with natural cranial suture variations, the limitations of this traditional localization method become apparent. This is especially true for the long-clawed gerbil—an ideal animal model for studying the neural regulation of social and aggressive behaviors. Its VMHvl nucleus is closely related to the regulation of instinctive behaviors such as aggression and defense, making it a hot research area in this field. However, this species is an outbred strain with high genetic diversity, and significant individual variations exist in cranial suture development.

[0004] However, this study's anatomical observation of the skulls of adult male long-clawed gerbils revealed six significant variations in their cranial sutures. Only type a exhibited a regular triangular configuration; the remaining types b / c / d / e / f all showed sagittal suture deviation from the midline or coronal suture concave towards the caudal side, resulting in a blurred, bifurcated, and non-unique location of the "coronal-sagittal suture intersection." Forcibly using the coronal-sagittal suture intersection as the anterior fontanelle would introduce a huge systematic error—experimental data showed that the average offset in the ML direction using the traditional method reached 155.31 ± 25.57 μm (P < 0.001), far exceeding the width of the VMHvl nucleus itself (approximately 200–300 μm), causing all downstream interventions (lesioning, optogenetics, electrophysiology) to completely miss their target.

[0005] Existing improved methods, such as the Blasik and Ferry methods, are mostly designed for mice and rats. The location markers they rely on, such as the "midpoint of the interauricular line" and the "base of the zygomatic suture triangle," are not visible on the skull of long-clawed gerbils, still requiring subjective judgment and failing to solve the fundamental problem. Therefore, there is an urgent need for a new location method that uses highly conserved anatomical points as a benchmark and allows for full quantification of instrument readings.

[0006] The VMHvl nucleus, a small and highly differentiated brain region, has different subregions that regulate various instinctive behaviors such as attack, defense, and mating. Therefore, its localization accuracy is far higher than that of other brain regions; even minor localization errors can lead to misjudgments of functional phenotypes. Currently, there are no precise VMHvl localization methods for animals with craniosynostosis variations, nor have spatial coordinates for the VMHvl nucleus been established to fit the anatomical characteristics of the long-clawed gerbil. This severely limits the progress of VMHvl functional research using the long-clawed gerbil as a model. Summary of the Invention

[0007] The purpose of this invention is to provide a VMHvl brain region localization method applicable to craniosynostosis variant animals, in order to solve the problems mentioned in the background art, such as the fact that the localization landmarks on which the method is relied upon are not visible in the actual skull and still require subjective judgment.

[0008] The above-mentioned objective of the present invention is achieved through the following technical solution: a method for locating the VMHvl brain region in animals with craniosynostosis variations, characterized by comprising the following steps: S1. Adult male long-clawed gerbils were anesthetized and fixed on a stereotaxic instrument to expose the skull surface, remove connective tissue, and clearly display the sagittal suture, coronal suture, and lambdoid suture. S2. Using the intersection of the sagittal suture and the herringbone suture as the posterior fontanelle, set the X, Y, and Z coordinates of the digital stereoscopic positioning instrument to zero, and control the positioning needle to move forward (towards the head end) along the Y-axis (anteroposterior direction) from the posterior fontanelle point at a constant speed. Stop when the needle tip reaches the visual / microscopic intersection of the coronal suture. This intersection is the anterior fontanelle. S3. Using the anterior fontanelle as a reference point, record its Z-axis reading (Z anterior fontanelle); then move the positioning pin back along the Y-axis to the posterior fontanelle point and record its Z-axis reading (Z posterior fontanelle); by adjusting the height of the incisor adapter, change its traction tension on the gerbil incisors, so that |Z anterior fontanelle - Z posterior fontanelle| ≤ 0.03 mm, confirming that the skull is successfully leveled in the anterior-posterior direction; move it 2 mm to the left and right, and record the Z-values ​​on the left and right sides respectively, so that |Z left - Z right| ≤ 0.05 mm, confirming that the skull is leveled in the left and right directions, and record the Z-values ​​of the anterior and posterior fontanelles and the left and right Z-values, and the morphology of the cranial sutures; S4. Using the anterior fontanelle as the origin of the coordinate system, take ±0.75 mm in the inner-outer (ML) direction, -1.7 mm in the anterior-posterior (AP) direction, and -7.5 mm in the dorsal-ventral (DV) direction as the needle insertion point for VMHvl. S5. At the skull marker drilling location, drill a hole with a 1 mm diameter drill bit to expose the dura mater and puncture it to stop bleeding. Then, insert the positioning needle to a depth of DV-7.5 mm and stop the needle for 1 minute to complete the positioning.

[0009] In this invention, the posterior suture is determined by opening the microscope display screen, finding the skull position, focusing, and then identifying the intersection of the lambdoid suture and the sagittal suture as the posterior suture. The cranial suture morphology was observed to be divided into six types: type a is a regular and standard type; types b, c, d, and f are irregular types where the sagittal suture deviates from the midline of the skull; type e is a type where the intersection of the coronal and sagittal sutures is concave towards the caudal side; only type a can construct a stable triangular configuration of sagittal suture-V-shaped suture-coronal suture, while the other five types cannot construct a stable triangular configuration of sagittal suture-V-shaped suture-coronal suture.

[0010] Preferably, the anterior fontanelle is positioned by using the posterior fontanelle as an anatomical reference and translating it forward along the Y-axis to the point where it intersects with the coronal suture.

[0011] As a preferred embodiment, the fixation method described in S1 includes: inserting a 60° angled ear rod into the gerbil's external ear canal at the horizontal level; after the ear rod is correctly fixed, aligning the animal's head with the center line of the stereoscopic positioning device; and using a mouse-specific incisor adapter, gently pulling it forward and tightening it for fixation.

[0012] As a preferred embodiment, when the mouse skull is leveled in the left-right direction as described in S3, if the error between the anterior fontanelle and posterior fontanelle is greater than 0.05 mm, the left and right ear rods need to be adjusted to re-fix the mouse brain, and the anterior and posterior fontanelles need to be re-fixed after adjustment.

[0013] As a preferred embodiment, the drilling described in S5 uses a 1 mm diameter drill bit, and the drill hole is only large enough for the positioning needle to pass through. Drilling is stopped when blood vessels are visible on the surface of the dura mater. A thin bone fragment is removed and the dura mater is punctured with a 1 mL syringe hook. After applying pressure with a cotton swab to stop the bleeding, the needle is inserted.

[0014] As a preferred method, the verification method for the VMHvl target coordinates described in S4 is as follows: after positioning, the head is severed and the brain is removed, a 90μm thick coronal frozen section is prepared, Nissl staining is performed, the morphology of the VMHvl nucleus is identified under an optical microscope, and the distance from the center of the needle path to the geometric center of VMHvl is measured.

[0015] Preferably, as verified by Nissl staining, the offset of the needle track in the ML direction is ≤5 μm, and in the AP direction it is within the range of -2.00 mm to -2.70 mm, and the overlap rate between the center of the needle track and the center of VMHvl is ≥95%.

[0016] Preferably, the VMHvl target coordinates (ML direction ±0.75mm, AP -1.7mm, DV -7.5mm) are allowed to fluctuate within the following ranges: ML direction ±0.10mm, AP direction ±0.15mm, and DV direction ±0.20mm.

[0017] As a preferred option, in S3, ImageView software is used to measure the distance from the needle track center to the geometric center of VMHvl, and this distance ≤ 5 μm is used as the positioning qualification criterion.

[0018] Preferably, the method is applicable to neural function studies that require destruction of VMHvl, electrophysiological recording, optogenetic manipulation, chemogenetic intervention, or viral tracing.

[0019] Preferably, the determination of the posterior fontanelle also includes any of the following auxiliary confirmation methods: (1) Under the surgical microscope, observe whether there is local bone thickening or vascular convergence feature point, with the intersection of the zygomatic suture and the sagittal suture as the center; (2) Combine preoperative miniature CT scan to reconstruct a three-dimensional model of the skull, and accurately mark the spatial intersection of the sagittal suture and the lambdoid suture in the model; (3) Use a laser positioning pen to project a cross line, align the intersection point with the visual intersection area of ​​the herringbone suture and the sagittal suture, and take the centroid coordinates of the three repeated positioning as the posterior fontanelle.

[0020] The beneficial effects of this invention are: For animals with cranial suture variations, such as the long-clawed gerbil, the anterior fontanelle is determined by quantitative translation using the posterior fontanelle as a reference and a digital display locator. This eliminates the reliance of traditional methods on the intersection of the sagittal and coronal sutures and fundamentally removes the systematic positioning errors caused by irregular cranial sutures.

[0021] The Z-axis height difference quantitative leveling (|Z anterior fontanelle - Z posterior fontanelle| ≤ 0.03 mm) is used to achieve objective calibration of the skull's horizontal state, avoiding anterior-posterior tilt errors caused by subjective judgment and significantly improving positioning consistency.

[0022] The precise coordinates and reasonable fluctuation range of VMHvl in long-clawed gerbils are given. Histological verification shows that the needle path offset is ≤5 μm and the nucleus overlap rate is ≥95%. The positioning accuracy is much higher than that of traditional methods, which meets the needs of precise intervention for small nuclei.

[0023] The localization process is standardized and repeatable, with instrument readings quantified throughout, eliminating reliance on operator experience. It is suitable for various neurofunctional experiments, including lesioning, electrophysiology, optogenetics / chemogenetics, and virus tracing. It reduces functional phenotypic errors caused by localization bias to a negligible level (P < 0.001), significantly improving the reliability and reproducibility of experimental data, and providing stable and reliable technical support for the study of VMHvl-related neural circuits in cranial suture variant animals. Attached Figure Description

[0024] Figure 1 A schematic diagram of the key cranial sutures on the surface of the skull of a long-clawed gerbil.

[0025] Figure 2This diagram illustrates six types of cranial suture variations in the skull of the long-clawed gerbil.

[0026] Among them: type a is the regular standard type, types b, c, d, and f are types where the sagittal suture deviates from the midline, and type e is the type where the intersection of the coronal suture and the sagittal suture is concave.

[0027] Figure 3 This is a statistical chart showing the offset of the ML direction in the localization of the VMHvl brain region between the example group and the comparative group.

[0028] Figure 4 This is a morphological diagram of the needle path for locating the VMHvl brain region in the example group.

[0029] Figure 5 This is a morphological diagram of the needle pathway in the VMHvl brain region of the comparative group. Detailed Implementation

[0030] The present invention will be further described in detail below with reference to the accompanying drawings.

[0031] This specific embodiment is merely an explanation of the present invention and is not intended to limit the invention. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they are within the scope of the claims of the present invention.

[0032] Materials and Methods 1. Materials 1.1 Laboratory Animals Seventeen male long-clawed gerbils, aged 70-90 days and weighing 60-80g, were obtained from the Zhejiang Provincial Experimental Animal Center and housed in the Zhejiang Provincial Key Laboratory of Vector Biology and Pathogen Control. The rearing temperature was 22 ± 2℃ and the relative humidity was 50% ± 5%. All procedures in this experiment complied with the guidelines for animal use and care of Huzhou University.

[0033] 1.2 Experimental Equipment and Reagents Standard digital stereotaxic instrument (68804, Shenzhen Ruiwode Co., Ltd.), cryostat (CM1850, Leica GmbH, Germany), surgical microscope (LZJ-6EL, Zhenjiang Zhongtian Optical Instrument Co., Ltd.), miniature handheld cranial drill (8001, Shenzhen Ruiwode Co., Ltd.), anti-detachment slides (Jiangsu Shitai Experimental Equipment Co., Ltd.), Nissl stain (C0117, Shanghai Beyotime Biotechnology Co., Ltd.), alcohol, xylene, etc.

[0034] 2 methods Seventeen male long-clawed gerbils were randomly divided into two groups. The control group (n=8) used the traditional anterior fontanelle definition method for VMHvl brain region localization. The example group (n=9) used a modified anterior fontanelle localization method (i.e., the VMHvl brain region localization method applicable to animals with craniosynostosis variations as described in this invention) for localization. The two groups of animals were kept in the same conditions, underwent anesthesia and fixation, underwent histological identification, and used the same statistical analysis methods. The only difference was in the anterior fontanelle determination and craniofacial leveling methods for nucleus localization, as detailed below: Example Disinfection and exposure of the surgical area: Disinfect the surgical area three times with povidone-iodine, make a longitudinal incision in the scalp and separate the subcutaneous tissue. Gently wipe the surface of the skull with a cotton swab soaked in 3% H2O2 to thoroughly remove connective tissue and bloodstains. Fully expose the sagittal suture, coronal suture and lambdoid suture on the surface of the skull, and carefully observe and record the morphology of the cranial sutures.

[0035] Determining the posterior and anterior fontanelles: First, identify the intersection of the sagittal suture and the lambdoid suture on the skull surface, and clearly define this intersection as the posterior fontanelle. Turn on the digital stereotactic instrument, align the tip of the positioning needle with the posterior fontanelle, and zero all X, Y, and Z axes of the stereotactic instrument. Control the positioning needle to move forward (towards the head) along the Y-axis from the posterior fontanelle at a uniform speed (0.1 mm / s). Observe the position of the needle tip in real time under the surgical microscope. When the needle tip reaches the visual / microscopic intersection of the coronal suture, stop moving immediately. This intersection is the anterior fontanelle (obtained by translating forward along the Y-axis to intersect with the coronal suture, using the posterior fontanelle as the anatomical reference).

[0036] Craniofacial leveling: Using the precisely determined anterior fontanelle as a reference point, gently touch the skull surface at the anterior fontanelle with the tip of the positioning needle, and record the Z-axis reading at this time (denoted as Z-anterior fontanelle). Then, control the positioning needle to move backward at a uniform speed along the Y-axis back to the posterior fontanelle point, and similarly gently touch the skull surface at the posterior fontanelle with the tip of the needle, and record the Z-axis reading at this time (denoted as Z-posterior fontanelle). By adjusting the height of the support rod fixing the gerbil's nose and changing the tension of the incisor adapter, after repeated fine adjustments, ensure that |Z-anterior fontanelle - Z-posterior fontanelle| ≤ 0.03 mm, confirming that the anterior-posterior horizontal leveling of the skull is successful. Then, verify the horizontal leveling of the skull by following the same method as the comparative example (the difference between the left and right Z-axis readings ≤ 0.05 mm), ensuring that the overall craniofacial leveling meets the standard.

[0037] Needle insertion points and burr insertion: Using the precisely determined anterior fontanelle as the origin of the coordinate system, the VMHvl insertion points are set at ±0.75mm in the medial-lateral (ML) direction, -1.7mm in the anterior-posterior (AP) direction, and -7.5mm in the dorsal-ventral (DV) direction (the allowable fluctuation range for this coordinate system is ±0.10mm in the ML direction, ±0.15mm in the AP direction, and ±0.20mm in the DV direction). Subsequent burr insertion, dura mater exposure, hemostasis, and needle insertion are performed in the same manner as the comparative model. That is, after marking the burr insertion points, a 1mm drill bit is used to drill a hole (the hole size is only large enough for the positioning needle to pass through, avoiding contact with the skull wall). After removing the thin bone fragment, puncturing the dura mater, and achieving hemostasis, the needle is inserted to a depth of DV-7.5mm, and the needle is stopped for 1 minute to complete the localization of the nucleus.

[0038] Comparative Example Disinfection and exposure of the surgical area: The surgical area of ​​the gerbil's head was thoroughly disinfected three times with povidone-iodine. The scalp tissue was longitudinally incised, and the subcutaneous tissue was separated with ophthalmic forceps. The surface of the skull was then gently wiped with a cotton swab soaked in 3% H2O2 to remove attached connective tissue and bloodstains, and the sagittal and coronal sutures on the surface of the skull were fully exposed. The morphology of the cranial sutures was recorded.

[0039] Anterior fontanelle determination: Using the traditional definition method, the visual intersection of the sagittal suture and the coronal suture on the surface of the skull is directly identified as the anterior fontanelle, and this point is used as the positioning reference point.

[0040] Craniofacial leveling: Using the previously determined anterior fontanelle as the origin, control the positioning needle to move 2mm left and right, lower the needle until the needle tip gently touches the skull surface, read the Z-axis reading at the corresponding position, adjust the height of the incisor adapter so that the difference between the Z-axis readings on the left and right sides is ≤0.05mm, and determine that the skull is horizontal from left to right; then move the positioning needle backward to the suture area (the approximate position determined subjectively), read the Z-axis reading, adjust the incisor adapter so that the difference between the Z-axis reading of the anterior fontanelle and the subjectively determined position is ≤0.02mm, and determine that the skull is horizontal from front to back.

[0041] Needle insertion points and drilling: Using the anterior fontanelle determined by the aforementioned traditional method as the origin of the coordinate system, the VMHvl insertion points were set at ±0.75mm in the medial-lateral (ML) direction, -1.7mm in the anterior-posterior (AP) direction, and -7.5mm in the dorsal-ventral (DV) direction. The drilling points were marked on the corresponding locations on the skull with a marker pen. A 1mm diameter drill bit was fitted to the miniature cranial drill, and a hole was drilled at the marked points. Drilling was stopped immediately when the meningeal vessels were faintly visible at the bottom of the hole. A thin bone fragment was picked out with a homemade 1ml syringe hook to expose the dura mater. After puncturing the dura mater in the same way, hemostasis was achieved by gently applying pressure with a sterile cotton swab. The stereotactic arm was controlled to insert the positioning needle to a depth of DV-7.5mm, and the needle was stopped for 1 minute to complete the localization of the nucleus.

[0042] After localization of the brain nuclei, the gerbils were removed from the stereotaxic apparatus, euthanized by cervical dislocation, and immediately decapitated to harvest the brain. The brain tissue was flash-frozen at -80°C for 30 minutes. After removal, it was transferred to a cryostat and thawed at -10°C for 20 minutes for 90 μm thick coronal sections. Brain sections were collected continuously, starting with the first needle mark. The brain sections were dried in a desiccator, fixed with 4% paraformaldehyde for 2 hours, and then perforated with PBST buffer at room temperature for 1 hour. They were then stained with Nissl stain at room temperature for 30 minutes. After staining, the sections were dehydrated sequentially with a gradient of alcohols (50%, 75%, 85%, 95%, 100%), cleared with xylene, and finally mounted with neutral resin. Brain slice images were acquired using an optical microscope and imported into ImageView software. The distance of the needle mark offset from the VMHvl brain region in the ML direction for each animal was measured and calculated. The degree of needle mark offset was analyzed to verify the localization accuracy.

[0043] Data analysis was performed using SPSS 22.0 software. All experimental data are expressed as mean ± standard deviation (x ± s). Normality was first tested. For data conforming to a normal distribution (e.g., needle mark offset distance in the ML direction), an independent samples t-test was used. For data not conforming to a normal distribution, a non-parametric test (Mann-Whitney U test) was used. A p-value < 0.05 was considered statistically significant. The differences in positioning accuracy between the control group and the control group were compared.

[0044] This experiment used 17 adult male long-clawed gerbils as research subjects, randomly divided into an example group (n=9, using the improved localization method of this invention) and a control group (n=8, using the traditional anterior fontanelle localization method). The accuracy and consistency of VMHvl brain region localization between the two groups were compared through Nissl staining histological identification, ImageView software image analysis, and statistical tests. Specific results are as follows: After localization, the brains were removed by decapitation, and coronal frozen sections and Nissl staining were performed. Observation under an optical microscope showed significant differences in the targeting accuracy of the needle tracks between the two groups: In the comparative group, the needle tracks of all 8 gerbils did not accurately target the VMHvl nucleus. Among them, the needle tracks of 5 gerbils deviated to the hypothalamic region adjacent to the medial or lateral side of the VMHvl nucleus, and the needle tracks of 3 gerbils penetrated the VMHvl nucleus to deep brain tissue. Moreover, the needle tracks were irregular in shape and scattered in distribution. In the example group, the needle tracks of all 9 gerbils accurately fell within the VMHvl nucleus. The needle tracks were regular in shape, consistent in direction, and corresponded well with the geometric center of the VMHvl nucleus, with no obvious deviation.

[0045] like Figure 3As shown, optical microscopy revealed that the needle tracks in the 8 gerbils in the comparative group did not precisely target the VMHvl nucleus and were scattered; in the example group, the needle tracks in all 9 gerbils were located within the VMHvl nucleus and had a regular morphology. In the comparative group, the needle track deviation of the cranial suture variant (bf type) gerbils was significantly greater than that of the standard type (a type), while the needle tracks of the cranial suture type gerbils in the example group could stably target the nucleus, effectively avoiding the deviation caused by cranial suture variants.

[0046] The average offset in the ML direction of the comparative example group was 155.31±25.57μm, while the average offset in the ML direction of the example group was only 2.29±0.96μm. The difference between the two groups was extremely significant (t=18.632, P<0.001).

[0047] The comparative group showed a discrete needle distribution along the AP direction, while the example group showed a highly concentrated needle distribution, resulting in better positioning consistency.

[0048] The pass rate was 0% for the control group and 100% for the example group, with a needle offset of ≤5μm as the standard. The average overlap rate between the needle track and the center of the VMHvl nucleus in the example group was 97.32±1.56% (all ≥95%), while the effective overlap rate could not be calculated for the control group.

[0049] In summary, this invention eliminates systematic errors caused by cranial suture variations, significantly improves the accuracy and consistency of VMHvl localization, and meets the needs of related neurological functional experiments. The VMHvl brain region localization method described in this invention, applicable to animals with cranial suture variations, effectively eliminates systematic errors caused by cranial suture variations, significantly improves the accuracy and consistency of localization, and achieves the required localization success rate and nucleus overlap rate for experimental research, thus meeting the needs of neurological functional experiments related to VMHvl nuclei.

Claims

1. A method for locating the VMHvl brain region in animals with cranial suture variations, characterized in that, Includes the following steps: S1. Adult male long-clawed gerbils were anesthetized and fixed on a stereotaxic instrument to expose the skull surface, remove connective tissue, and clearly display the sagittal suture, coronal suture, and lambdoid suture. S2. Using the intersection of the sagittal suture and the herringbone suture as the posterior fontanelle, set the X, Y, and Z coordinates of the digital stereoscopic positioning instrument to zero, and control the positioning needle to move forward (towards the head end) along the Y-axis (anteroposterior direction) from the posterior fontanelle point at a constant speed. Stop when the needle tip reaches the visual / microscopic intersection of the coronal suture. This intersection is the anterior fontanelle. S3. Using the anterior fontanelle as a reference point, record its Z-axis reading (Z anterior fontanelle); then move the positioning pin back along the Y-axis to the posterior fontanelle point and record its Z-axis reading (Z posterior fontanelle); by adjusting the height of the incisor adapter, change its traction tension on the gerbil incisors, so that |Z anterior fontanelle - Z posterior fontanelle| ≤ 0.03 mm, confirming that the skull is successfully leveled in the anterior-posterior direction; move it 2 mm to the left and right, and record the Z-values ​​on the left and right sides respectively, so that |Z left - Z right| ≤ 0.05 mm, confirming that the skull is leveled in the left and right directions, and record the Z-values ​​of the anterior and posterior fontanelles and the left and right Z-values, as well as the morphology of the cranial sutures; S4. Using the anterior fontanelle as the origin of the coordinate system, take ±0.75 mm in the inner-outer (ML) direction, -1.7 mm in the anterior-posterior (AP) direction, and -7.5 mm in the dorsal-ventral (DV) direction as the needle insertion point for VMHvl. S5. At the skull marker drilling location, drill a hole with a 1 mm diameter drill bit to expose the dura mater and puncture it to stop bleeding. Then, insert the positioning needle to a depth of DV-7.5 mm and stop the needle for 1 minute to complete the positioning.

2. The VMHvl brain region localization method applicable to craniosynostosis variant animals according to claim 1, characterized in that, The anterior fontanelle is located by using the posterior fontanelle as an anatomical reference and translating it forward along the Y-axis to the point where it intersects with the coronal suture.

3. The VMHvl brain region localization method applicable to craniosynostosis variant animals according to claim 1, characterized in that, The fixation method described in S1 includes: inserting a 60° angled ear rod into the gerbil's external ear canal at the horizontal level; after the ear rod is correctly fixed, aligning the animal's head with the center line of the stereoscopic positioning device; and using a mouse-specific incisor adapter, gently pulling it forward and tightening it for fixation.

4. The VMHvl brain region localization method applicable to craniosynostosis variant animals according to claim 1, characterized in that, If the error between the anterior and posterior fontanelles is greater than 0.05 mm when the left and right sides of the mouse skull are adjusted as described in S3, the mouse brain needs to be re-fixed by adjusting the left and right ear rods. After adjustment, the anterior and posterior fontanelles need to be re-fixed.

5. The VMHvl brain region localization method applicable to craniosynostosis variant animals according to claim 1, characterized in that, The drilling described in S5 uses a 1 mm diameter drill bit, and the hole size is only large enough for the positioning needle to pass through. Drilling is stopped when blood vessels are visible on the surface of the dura mater. A thin bone fragment is removed and the dura mater is punctured with a 1 mL syringe hook. After applying pressure with a cotton swab to stop the bleeding, the needle is inserted.

6. The VMHvl brain region localization method applicable to craniosynostosis variant animals according to claim 1, characterized in that, The verification method for the VMHvl target coordinates described in S4 is as follows: After positioning, the head is severed and the brain is removed. A 90 μm thick coronal frozen section is prepared, Nissl staining is performed, the morphology of the VMHvl nucleus is identified under an optical microscope, and the distance from the center of the needle path to the geometric center of VMHvl is measured.

7. The VMHvl brain region localization method applicable to craniosynostosis variant animals according to claim 3, characterized in that, Verified by Nissl staining, the offset of the needle track in the ML direction is ≤5 μm, and in the AP direction it is within the range of -2.00 mm to -2.70 mm, and the overlap rate between the center of the needle track and the center of VMHvl is ≥95%.

8. The VMHvl brain region localization method applicable to craniosynostosis variant animals according to claim 1, characterized in that, The VMHvl target coordinates (ML direction ±0.75mm, AP -1.7mm, DV -7.5mm) are allowed to fluctuate within the following ranges: ML direction ±0.10mm, AP direction ±0.15mm, DV direction ±0.20mm.

9. The VMHvl brain region localization method applicable to craniosynostosis variant animals according to claim 1, characterized in that, In S3, ImageView software is used to measure the distance from the needle track center to the geometric center of VMHvl, and this distance ≤5 μm is used as the positioning qualification criterion.

10. The VMHvl brain region localization method applicable to craniosynostosis variant animals according to any one of claims 1 to 8, characterized in that, This method is applicable to neural function studies that require lesioning of VMHvl, electrophysiological recording, optogenetic manipulation, chemogenetic intervention, or viral tracing.

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