DISPOSITIVO DE ESTIMULAÇÃO CEREBRAL PROFUNDA EM ESCALA REDUZIDA PARA NEUROMODULAÇÃO INVASIVA EM CAMUNDONGOS

Description

/ 20 Small-scale deep brain stimulation device for invasive neuromodulation in mice. FIELD OF THE INVENTION

[001] The present invention is situated in the field of deep brain stimulation devices and invasive neuromodulation devices and relates to the use for studying the neuroprotective and antiparkinsonian effect of this device in animal models. The device consists of intracerebral electrodes coupled to a small printed circuit board, suitable for implantation in the brain of small rodents, such as mice. STATE OF THE ART

[002] Neuromodulation, especially through deep brain stimulation (DBS), is widely used in humans for the treatment of neurodegenerative diseases, such as Parkinson's disease (PD). However, there are significant differences between devices designed for human use and those developed for preclinical research in animal models, such as mice.

[003] Several patents and research papers describe relevant technologies, but with approaches distinct from the proposal of this device. Below, we present a table with significant patents and their purposes: Petition 870250004911, dated 22 / 01 / 2025, page 8 / 92 / 20

[004] Table 1 - Main patent documents Patent Number Publication Date Main Classification Purpose 1 US11833 347B2 06 / 19 / 2015 A61N1 / 0534 Miniaturization of components and integration of stimulation systems with real-time monitoring for human use. 2 US11167 138B2 06 / 19 / 2015 A61N1 / 0534 Increasing cerebral blood flow in humans, without focus on neuromodulation of deep brain nuclei. 3 US2013 / 0281890 A1 11 / 11 / 2009 A61N7 / 00 Neuromodulation combined with additional therapies, such as thermal stimulation. 4 US2017 / 0291044 A1 10 / 12 / 2017 A61N7 / 02 Non-invasive neuromodulation using ultrasound to alter brain activity. 5 BR 11 2023 025454 0 28 / 05 / 2022 A61N 1 / 05 Subgaleal implant for cortical and / or deep brain stimulation and / or modulation in humans. 6 BR 11 2017 006509 6 24 / 07 / 2015 A61N 1 / 05 Deep brain stimulation through implantation and activation of Petition 870250004911, dated 01 / 22 / 2025, page 9 / 92 / 20 brain conductors. 7 BR 10 2013 000663 7 10 / 01 / 2013 A61B 5 / 0478 Closed-loop deep brain stimulation with external control, with a feedback system that modulates the electrical stimuli of the deep brain electrode as a function of cerebral cortical activity in humans. 8 CN11605 9531A CN11543 0049A 05 / 05 / 2023 06 / 10 / 2022 A61B5 / 00 Device for recording neuronal activity frequency and electrical neuromodulation in an animal model of Alzheimer's disease in mice. 9 US20172 46481A1 31 / 08 / 2017 A61N7 / 00 Technology that proposes the use of ultrasound to neuromodulate brain structures in humans. 10 US11154 715B2 US20193 21626A1 24 / 10 / 2019 26 / 10 / 2016 A61N1 / 05 Deep brain stimulation device that neuromodulates the midbrain of rats that have suffered cerebral ischemia, to improve their gait performance. 11 US20141 72045A1 19 / 06 / 2014 23 / 05 / 2017 A61N1 / 372 Automation system for Petition 870250004911, dated 01 / 22 / 2025, page 10 / 92 / 20 US96560 89B2 Parameter adjustments for deep brain stimulation in humans. 12 CA28575 55A1 01 / 10 / 2015 A61N1 / 36 Non-invasive neuromodulation in humans using electromagnetic pulses. 13 WO20240 73597A1 04 / 04 / 2024 A61B5 / 00 System for optimizing neurostimulation therapies, whether electrical, magnetic or of another nature. 14 US20213 69394A1 02 / 12 / 2021 A61B34 / 10 Artificial intelligence-based ecosystem that can assist in decision-making in the programming of neuromodulation devices in humans. 15 CA29176 54A1 CA29176 54C 14 / 07 / 2017 25 / 09 / 2018 A61B34 / 20 Neuro-navigation system for targeting deep brain nuclei more accurately.

[005] We have that in the patent databases there is a record of different technologies that address neuromodulation. Most of them deal with technologies developed to be used on a human scale. Others use not electricity, but ultrasound, electromagnetism and others.

[006] There are four studies that deal with small-scale invasive neuromodulation devices for use in preclinical research using mice (CN116059531A, CN115430049A, US11154715B2 and US2019321626A1). The first two Petition 870250004911, dated 01 / 22 / 2025, page 11 / 92 5 / 20 were developed with the aim of being used in pre-clinical research on Alzheimer's disease, and the other two in cerebral ischemia. Therefore, no devices with the purpose presented in this patent application are listed in the patent databases.

[007] Furthermore, the technologies mentioned above do not refer to the electrode's ability to deliver a bipolar electrical stimulation pulse, nor to features that facilitate its attachment to the animals' skull, reducing the risk of displacement.

[008] Regarding non-patent bases, some research centers studying invasive neuromodulation in mice use electrodes manufactured by companies (Plastics One (https: / / www.pltec.com / ). This company is a supplier of electrodes and other components for neuromodulation, and its technology has been used in some research (Lowet et al. Deep brain stimulation creates informational lesion through membrane depolarization in mouse hippocampus 13:7709, 2022; Schmuckermair et al. Behavioral and neurobiological effects of deep brain stimulation in a mouse model of high anxiety-and depression-like behavior. 38:1234-1244, 2013; Mann et al. Chronic deep brain stimulation in an Alzheimer's disease mouse model enhances memory and reduces pathological hallmarks. 11:435-444, 2018).

[009] The electrode offered by Plastics One consists of two monopolar electrodes coupled to a device that assists in attaching the electrodes to the mouse brain and has female connectors for attaching them to an external electrical stimulator.

[0010] Another company is FHC (https: / / www.fh-co.com / ) which also supplies electrodes and stimulation systems. Relevant studies include: Halpern et al. A step-wise approach to deep brain Petition 870250004911, dated 01 / 22 / 2025, page 12 / 92 / 20 stimulation in mice. 156:1515-1521, 2024; Valverde et al. Deep brain stimulation-guided optogenetic rescue of parkinsonian symptoms. 11:1-17, 2020.

[0011] The device offered by FHC is bipolar, but there is no mention of features that facilitate its attachment to the animal's skull.

[0012] Other laboratories manufacture their own electrodes manually, resulting in devices with varying characteristics in length, weight, diameter of the stimulation area, materials, and polarity (Hao et al. Forniceal deep brain stimulation rescues hippocampal memory in Rett syndrome mice. 526:430-434, 2015; Melo-Thomas et al. Electrical stimulation or MK-801 in the inferior colliculus improves motor deficits in MPTPtrea ted mice. 65:38-43, 2018; Schor et al. Multiple stimulation parameters influence efficacy of deep brain stimulation in parkinsonian mice.1129: 3833 - 3838, 2019; Van den Boom et al. Unraveling the mechanisms of deep-brain stimulation of the internal capsule in a mouse model. 14:1-17, 2023).

[0013] The present invention relates to a deep brain stimulation (DBS) system developed for use in rodents, particularly in experimental models of neurodegenerative diseases such as Parkinson's disease. The device consists of intracerebral electrodes coupled to a small printed circuit board, suitable for stereotaxis implantation in the brains of small rodents such as mice.

[0014] The device can be used in neuroscientific studies to evaluate the effects of deep brain stimulation on neuroprotection and motor behavior in rodents, representing a significant advance in experimental models for the study of diseases such as Parkinson's disease. Petition 870250004911, dated 22 / 01 / 2025, page 13 / 92 / 20

[0015] The invention utilizes an intracerebral electrode designed for neuromodulation in specific brain regions, such as the Subthalamic Nucleus (STN), assisting in the evaluation of motor and neuroprotective responses.

[0016] This system is designed to be minimally invasive, with small electrodes that can be precisely implanted using stereotactic techniques. The electrode design facilitates its insertion into deep brain targets, such as the NST, and its efficient operation in experimental biomedical research settings.

[0017] The innovation of the invention lies in the integration of the electrode with a plate designed to optimize the positioning and anchoring of the electrode in order to achieve precise electrical stimulation, capable of generating safe neuromodulation of brain regions of interest. This feature is ideal for ensuring the effectiveness of deep brain stimulation under rigorous experimental conditions, enabling studies with the highest scientific standards.

[0018] Another distinguishing feature of the system is its ability to be used in conjunction with other recording techniques, such as behavioral and neuroimaging studies, in research protocols involving the evaluation of neuroprotection in injury-induced models, such as the injection of 6-OHDA neurotoxin, common in research on Parkinson's disease.

[0019] The system was designed primarily for experiments with small rodents, such as mice, allowing precise stimulation of regions such as the NST of these animals.

[0020] Through this technology, it is possible to conduct studies on the modulation of brain activity and its effects on motor behavior, focusing on the identification of potentials. Petition 870250004911, dated 01 / 22 / 2025, page 14 / 92 / 20 neuroprotective effects.

[0021] The invention can be used in combination with a wide range of commercial stimulators, since its generic connectors allow for easy coupling or adaptation to these devices. This versatility allows the system to be integrated with different stimulation equipment available on the market, making it a practical and flexible tool for neuroscience research, as well as expanding its applications in various experimental contexts.

[0022] It is also worth noting that the invention is not limited to the examples described here, and can be adapted to different experimental contexts involving neuromodulation in small animal species, expanding its applications in biomedical research and experimental therapies.

[0023] In short, there are neuromodulation devices of different natures, both in patent and non-patent databases. Most of them were developed for use on a human scale. Others use energy from electromagnetic pulses, ultrasound, and other sources in addition to electrical energy. There is no single device that combines the characteristics of being standardized, delivering bipolar electrical pulses, properly attaching to the animal's brain, reducing the risk of electrode displacement from the intracerebral target, reducing the risk of an inadequate connection between the device and the external power source, facilitating its coupling to stereotactic devices, and being very versatile in coupling to different external stimulation systems.

[0024] Furthermore, regarding costs, the prices of currently marketed intracerebral electrodes fluctuate. Petition 870250004911, dated 01 / 22 / 2025, page 15 / 92 9 / 20 between 30 and 50 dollars (https: / / protechinternational.com / products / 2channel-electrode-ms303-9-b-spc-twisted-platinum; (https: / / protechinternational.com / products / 3-channel-electrode-ms333-6-a-spcplatinum-iridium) while the developed neuromodulation device had an estimated cost of less than six dollars per unit, making the technology more accessible. BRIEF DESCRIPTION OF THE FIGURES

[0025] The invention can be better understood based on Figures 1 to 9, the description of which follows below:

[0026] Figure 1 shows the 3D design of the deep brain stimulation (DBS) system developed for use in rodents.

[0027] Figure 2 shows the layout of the printed circuit board that was designed to receive the electrode and connectors of the stimulator.

[0028] Figure 3 shows a top view of the printed circuit board, where the gold elements indicate the copper solder points.

[0029] Figure 4 shows the bottom view of the printed circuit board, where the gold elements indicate the copper solder points and the light yellow lines, the conductive traces.

[0030] Figure 5 is a schematic representation of the experimental groups used in the research where the neuromodulation device was tested, a) 6-OHDA + DBS Group, b) 6OHDA Group and c) Naive.

[0031] Figure 6 shows the data on changes in body mass, measured in grams, over time in these three experimental groups, analyzed using the Two-Way ANOVA statistical test. Note: * Statistically significant difference between the 60HDA and Naive groups with p<0.05. Petition 870250004911, dated 01 / 22 / 2025, page 16 / 92 / 20

[0032] Figure 7 shows the dwell time data during the Rotarod test for the 6-OHDA + DBS and 6-OHDA groups, analyzed using the Mann-Whitney statistical test. Note: * p<0.05.

[0033] Figure 8 shows the skewness coefficient data for the three experimental groups, analyzed using a two-way ANOVA statistical test. Note: * Statistically significant difference between the 6-OHDA and Naive groups, p<0.05.

[0034] Figure 9 shows the data from the dopaminergic cell counts obtained from micrographs in the right SN of animals in the 6-OHDA + DBS experimental group (6OHDA + DBS SN D), in the left SN of animals in the 6-OHDA + DBS experimental group (6OHDA + DBS SN E), in the right SN of animals in the 6-OHDA experimental group (6OHDA SN D), and in the left SN of animals in the 6-OHDA experimental group (6OHDA SN E), analyzed using the One-Way ANOVA statistical test with Welch's correction. Note: *: p<0.05, **: p<0.01, *** p<0.001.

[0035] Figure 10 shows the optical density (OD) data of the right striatum of the 6-OHDA+DBS experimental group (6OHDA + DBS D) and the left striatum of the 6-OHDA+DBS experimental group (6OHDA + DBS E), analyzed using the paired Student's t-test.

[0036] Figure 11 shows the OD data of the right striatum of the 6-OHDA experimental group (6OHDA D) and the left striatum of the 6-OHDA experimental group (6OHDA E), analyzed by the paired Student's t-test. Note: **p<0.01. DETAILED DESCRIPTION OF THE INVENTION

[0037] The intracerebral electrode, as shown in Figure 1, is manufactured using a blunt-tipped needle made of surgical steel, 13 mm long and 26G gauge. The needle acts as the cathode and houses an insulated platinum / iridium wire with 0.14 Petition 870250004911, dated 01 / 22 / 2025, page 17 / 92 / 20 mm in diameter that acts as an anode. This electrode is connected to a specialized connector, which facilitates communication with the external electrical stimulator, allowing the modulation of specific brain areas, such as the NST, which is involved in neurodegenerative processes, in addition to serving as an extension of the device that facilitates its coupling to the arms of stereotactic devices.

[0038] The needle, in addition to being one of the electrode poles, acts as a guide so that the fragile platinum / iridium wire does not deform during its passage through the animal's brain during implantation.

[0039] The device also includes a two-layer printed circuit board (Figures 2, 3 and 4), made of insulating material (FR-4 fiberglass), 1.6 mm thick and 7x9 mm wide and long. The circuit is divided into two segments: the first contains the components that deliver the electrical stimulation (electrode) and the second contains the connector pins, which interface with the external stimulator.

[0040] To ensure accurate stimulation and avoid interference, the electronic components are encapsulated in an epoxy resin, which also protects the device from cerebrospinal fluid and potential external electromagnetic interference. The device's geometry was optimized to minimize invasiveness and impact on the areas surrounding the implanted electrode, thus minimizing damage to the animals' brain tissue.

[0041] On the side contralateral to the electrode, the system has a slot 3mm long by 1mm wide. This slot allows for balancing and anchoring of the system by inserting micro-screws that secure the system to the rodent's skull. The Petition 870250004911, dated 01 / 22 / 2025, page 18 / 92 / 20 The microscrew has a head diameter of 1.4 mm and a thread diameter of 1.0 mm, with a total length of 2.6 mm. This layout allows for safer deep stimulation, reducing the risk of electrode displacement.

[0042] The research protocol in which the small-scale deep brain stimulation device for invasive neuromodulation was tested used the mouse as an experimental platform to study the effects of early deep brain stimulation on induced parkinsonism in these animals. Parkinsonism was induced by intracerebral injection of the neurotoxin 6-hydroxydopamine (6-OHDA) and, at the same surgical time, the deep brain stimulation device was implanted, with the electrode tip at the following stereotaxic coordinates in relation to the bregma line of the mouse skull: -2.0 mm (anteroposterior), -1.5 mm (laterolateral) and -4.5 mm (dorsoventral) (Pol et al. A Custom Made Electrode Construct and Reliable Implantation Method That Allows for Long-Term Bilateral Deep Brain Stimulation in Mice. 24:212-219, 2021).

[0043] Male Swiss mice (20 to 35g) were used, acquired from the Animal Facility of the Institute of Biological Sciences of the University of Brasília, after approval by the ethics committee on the use of animals (CEUA). The experimental animals were housed two per cage and kept in an animal facility with a 12 / 12 hour light / dark cycle, controlled temperature (25 °C) and humidity (55%). Throughout the experimental period, water and food were offered ad libitum.

[0044] The protocol defined three experimental groups, through simple randomization (Figure 5): Petition 870250004911, dated 22 / 01 / 2025, page 19 / 92 / 20

[0045] Group 1: DBS implants, activated, and left nigrostriatal lesion (6-OHDA + DBS), n = 20. Eight animals died during the surgical procedure or during the protocol, n=12. After histological confirmation of correct electrode implantation, n = 6;

[0046] Group 2 (6-OHDA): DBS implants, inactive, and left nigrostriatal lesion (6-OHDA), n = 19. Five animals died during the surgical procedure or during the protocol, n=14. After histological confirmation of correct electrode implantation, n = 8;

[0047] Group 3 (Naive): No DBS implant and no nigrostriatal lesion, n = 5.

[0048] The protocol defined high-frequency deep brain stimulation using constant current monophasic square waves (130 Hz, 60 ps, ​​100 μA) using the S88 stimulator, Grass Telefactor, Warwick, RI, USA, for a period of 3 hours per day, for four days, always at the same time, between 2 pm and 5 pm, starting on the first day (D1) after surgery. 30G gauge wires were used to connect the stimulator to the deep brain stimulation device previously fixed to the animals' skulls. The wires were kept at a height of 30 cm and were held by a pedestal, without restricting the animals' movement.

[0049] Since the stimulator emits a constant voltage, to verify the amount of current delivered intracerebrally to the animal, a circuit connected to a Fnirsi DSO-TC3 oscilloscope was constructed, coupled to a P6100 probe, which allowed calculating the resistance of the circuit system of the intracerebral electrode and the mouse brain in the NST region, in ohms (R2). Petition 870250004911, dated 01 / 22 / 2025, p. 20 / 92 / 20

[0050] Using Ohm's law (Current: Voltage / Resistance), knowing the resistance values, the voltage, in millivolts, needed to deliver the desired current to the system was defined, in addition to ensuring that the brain stimulus was effectively reaching the target.

[0051] These stimulation parameters were chosen because they conform to safety standards for current density, absence of motor abnormalities per stimulus, and similarity to typical DBS settings used in humans (Montgomery. Deep Brain Stimulation Programming. Book: 131-141, 2016).

[0052] The animals' body mass was assessed from the day of surgery (D0) to the day of euthanasia (D7), and to quantify the effect of stimulation on motor changes caused by 6-OHDA injury, two behavioral tests were performed: the asymmetry test using a cylinder during brain electrical stimulation, and shortly after the fourth day (D4) of stimulation, the motor performance test using the Rotarod. An immunohistochemical study with Tyrosine Hydroxylase (TH) staining was also performed in the Substantia Nigra (SN) and striatum to quantify the difference in the number of nigral dopaminergic neurons, as well as the density of striatal dopaminergic terminals between the lesioned and intact sides. EXAMPLES OF CONCRETIZATIONS OF THE INVENTION

[0053] Application examples are presented here in order to illustrate certain embodiments of the invention in more detail. It is important to note that the present invention is not limited to the examples cited and can be used in all the applications described or in any other equivalent variations. Petition 870250004911, dated 22 / 01 / 2025, p. 21 / 92 / 20

[0054] Body mass change data were analyzed in the three experimental groups. After an initial statistical analysis of the data using the Shapiro-Wilk test, it was identified that the change data followed a normal distribution. A two-way ANOVA test was applied. Upon identifying an epsilon of 0.3025, the Geisser-Greenhouse correction was applied. It was identified that there was a change in body mass as a function of time in the animals [F (1,513, 24,2) = 6.044, p=0.01]. There was also a change in body mass associated with the interaction between treatment and time. [F (10, 80) = 4.723, p <0.001]. It was observed that there was a difference in the body mass of the animals as a function of the individuals [F (16, 80) = 40.91, p <0.001]. There was no difference in body mass as a function of the intervention performed on the injured animals [F (2, 16) = 1.883, p= 0.18].

[0055] The body mass of the animals in the 6-OHDA + DBS group remained the same as that of the Naive group throughout the protocol. The animals in the 6-OHDA group were different from the Naive group, both on D4 (p= 0.04) and on D7 (p= 0.03).

[0056] Table 2 of the percentage difference in body mass over time of the 6-OHDA + DBS, 6-OHDA and Naive groups Table 2 - Percentage difference in body mass over time Body mass D1-D0 (%) Body mass D2-D1 (%) Body mass D3-D2 (%) Body mass D4-D3 (%) Body mass D7-D4 (%) Naive + 0.62 + 0.85 +2.47 + 1.23 +3.7 6OHDA + DBS -9.38 0.0 + 1.72 + 1.69 + 6.66 6OHDA -9.86 -6.25 0.0 -5.0 + 1.75 Petition 870250004911, dated 01 / 22 / 2025, page 22 / 92 / 20

[0057] Thus, the animals in the 6-OHDA group, after the intervention, progressively lost body mass until D4, when the mass loss curve stabilized. In contrast, the animals in the 6-OHDA+DBS group began to show a recovery in body mass after D3. The animals in the 6OHDA + DBS group maintained a body mass equal to that of the Naive group, while the animals in the 6-OHDA group showed a lower body mass than the Naive group after D3 (Figure 6).

[0058] Regarding the animals' performance in the Rotarod test, the difference between the 6-OHDA + DBS group and the 6-OHDA group's Rotarod time data did not follow a normal distribution, as indicated by the Shapiro-Wilk test (p = 0.03). Therefore, the comparison between the 6-OHDA + DBS and 6-OHDA groups was made using the Mann-Whitney test. In this analysis, the U statistic of 5 and a p-value of 0.02 indicated that the 6-OHDA + DBS group performed better on the Rotarod than the 6-OHDA group.

[0059] Thus, it was identified that the animals in the 6-OHDA + DBS group showed superior motor performance when compared to those in the 6-OHDA group (Figure 7).

[0060] Regarding the animals' performance in the skewness coefficient measured using the cylinder test, the data followed a normal distribution, verified by the Shapiro-Wilk test. Therefore, a two-way ANOVA statistical test was used, with Geisser-Greenhouse correction, calculating an epsilon of 0.4892. There was a statistically significant difference in the skewness coefficient, as a function of the intervention performed on the animals in the three groups [F (2, 16) = 4.295, p= 0.03]. Petition 870250004911, dated 01 / 22 / 2025, page 23 / 92 / 20

[0061] However, with respect to time, there was no significant difference in the skewness coefficient between the three groups [F (1,467, 16,63) = 0.074, p=0.88]. Furthermore, when evaluating the interaction between treatment and time, there was also no significant change in the skewness coefficient in the three groups [F (6, 34) = 0.7686, p= 0.6].

[0062] In multiple comparisons between the groups, it was identified that the animals in the 6-OHDA group presented a statistically different skewness coefficient from the Naive group on D2 (p= 0.03) and D4 (p= 0.02). On the other hand, the animals in the 6-OHDA + DBS group did not show relevant differences in relation to the Naive group.

[0063] Thus, only the animals in the 6-OHDA group, unlike those in the 6-OHDA + DBS group, showed high and different skewness coefficients from those in the Naive group on days D2 and D4 (Figure 8).

[0064] Regarding the dopaminergic neuron count data in the SN, two subgroups were created from the 6-OHDA+ DBS group, as well as from the 6-OHDA group, totaling four subgroups: a) number of TH+ cells in the right CNS of animals in the group 6-OHDA + DBS (6OHDA + DBS SN D); b) number of TH+ cells in the left SN of animals in the group 6-OHDA + DBS (6OHDA + DBS SN E); c) number of TH+ cells in the right SN of animals in the group 6-OHDA (6OHDA SN D); d) number of TH+ cells in the left SN of animals in the group 6-OHDA (6OHDA SN E). Petition 870250004911, dated 01 / 22 / 2025, page 24 / 92 / 20

[0065] The normality of the TH+ cell count data in the groups was confirmed by the Shapiro-Wilk test. Variances were assessed by Bartlett's test, which showed statistically significant differences between them (p= 0.006). Thus, a One-Way ANOVA test with Welch's correction was used, which revealed a significant difference between the groups [F (3, 33, 82) = 10.51, p< 0.001].

[0066] In comparing the differences between the four subgroups (6OHDA + DBS SN D, 6OHDA + DBS SN E, 6OHDA SN D and 6OHDA SN E), it was observed that the TH+ cell count on the left side of the group of parkinsonian animals that did not receive neuromodulation (6OHDA SN E) corresponded to: - A value of 27.32% of TH+ cell count in the left SN of parkinsonian animals that received neuromodulation (6OHDA + DBS SN E) - A value of 51.68% of TH+ cell count in the right SN of parkinsonian animals that received neuromodulation (6OHDA + DBS SN D) - A value of 53.94% of the TH+ cell count in the right SN of parkinsonian animals that did not receive neuromodulation (6OHDA + SN D).

[0067] Table 3 shows the differences between the numbers of dopaminergic cells counted in the micrographs in the right SN of animals in the 6-OHDA + DBS group (6OHDA + DBS SN D), in the left SN of animals in the 6-OHDA + DBS group (6OHDA + DBS SN E), in the right SN of animals in the 6-OHDA group (6OHDA SN D) and in the left SN of animals in the 6-OHDA group (6OHDA SN E), analyzed by the One-Way ANOVA test with Welch correction. Petition 870250004911, dated 01 / 22 / 2025, page 25 / 92 / 20 Table 3 - Differences between the numbers of dopaminergic cells Welch — ANOVA 6OHDA + DBS SN D 6OHDA + DBS SN E 6OHDA SN D 6OHDA SN E 6OHDA + DBS SN D — Not different (p=0.18) Not different (p=0.91) Different (p=0.002) 6OHDA + DBS SN E Not different (p=0.18) — Not different (p=0.08) Different (p=0.03) 6OHDA SN D Not different (p=0.91) Not different (p=0.08) — Different (p<0.001) 6OHDA SN E Different (p=0.002) Different (p=0.03) Different (p<0.001) —

[0068] Thus, only parkinsonian animals that were not subjected to neuromodulation showed a lower number of dopaminergic cells in the SN ipsilateral to the nigrostriatal lesion, when compared to the contralateral side (Figure 9).

[0069] With the aim of comparing the density values ​​of striatal dopaminergic terminals between the lesioned side and the intact side in parkinsonian animals, the optical density (OD) of TH labeling was measured in micrographs of histological sections of the striatum of animals from the 6-OHDA+DBS and 6OHDA groups.

[0070] The Shapiro-Wilk test confirmed the normality of the data. OD data from both the left and right striatum were collected from both the 6OHDA+DBS and 6-OHDA groups. Subsequently, OD data from micrographs of the striatum were analyzed. Petition 870250004911, dated 01 / 22 / 2025, p. 26 / 92 / 20 right and left of the 6-OHDA+DBS group by the paired Student's t-test, which showed no difference between them (t = 0.9394, df = 5, p = 0.39). However, the same procedure on the OD data of the 6-OHDA group led to the identification of a significant difference between them (t = 3.601, df = 7, p = 0.009).

[0071] In summary, in the animals of the 6-OHDA+DBS group, the OD in the left hemisphere was 1% lower than in the right hemisphere, without statistical significance (p = 0.39). In the 6-OHDA group, the OD on the left side was 27% lower than on the right, a statistically significant difference (p = 0.009) (Figures 10 and 11). Petition 870250004911, dated 01 / 22 / 2025, p. 27 / 92

Claims

1 / 2 CLAIMS 1. A small-scale deep brain stimulation device characterized by generating bipolar pulses and having the ability to properly attach to the mouse brain.

2. BRAIN STIMULATION DEVICE, according to claim 1, characterized by being composed of: A) An intracerebral electrode, manufactured using a blunt-tipped needle made of surgical steel, 13 mm long and 26G gauge; B) The needle acts as a cathode and houses an insulated platinum / iridium wire with a diameter of 0.14 mm that acts as an anode; C) The electrode is connected to a specialized connector, which facilitates communication with the external electrical stimulator, allowing the modulation of specific brain areas, such as the Subthalamic Nucleus (STN), which is involved in neurodegenerative processes, in addition to serving as an extension of the device that facilitates its coupling to the arms of stereotactic devices; D) The electronic components are encapsulated in an epoxy resin. E) On the side opposite the electrode, the system has a slot 3mm long by 1mm wide. Petition 870250004911, dated 01 / 22 / 2025, page 1.28 / 92 2 / 2 F) The micro screw has a head diameter of 1.4 mm and a thread diameter of 1.0 mm, with a total length of 2.6 mm.

3. BRAIN STIMULATION DEVICE, according to claims 1 to 2, characterized by being properly attached to the animal's brain and capable of reducing the risks of electrode displacement from the intracerebral target, reducing the risks of an inadequate connection between the device and the external power source, facilitating its coupling to stereotactic devices, and being versatile in coupling to different external stimulation systems.

4. USE OF A STIMULATION DEVICE, according to claims 1 to 3, characterized by being for the study of invasive neuromodulation in a mouse model of Parkinson's disease.

5. USE OF A BRAIN STIMULATION DEVICE, according to claims 1 to 3, characterized by showing effects in reducing motor and neuroprotective alterations in a mouse model of Parkinson's disease. Petition 870250004911, dated 01 / 22 / 2025, p. 29 / 92

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