Analysis of hippocampus in rats with acute brain ischemia-reperfusion injury treated with leuprolide acetate,an agonist of GnRH

Abstract

Background:

The hippocampus is highly vulnerable to damage in the brain ischemia-reperfusion injury model. Leuprolide acetate has been shown to promote neurological recovery after injury in various regions of the central nervous system.

Objective:

The objective of this study was to assess the histology of the hippocampus and the expression of neuronal recovery markers, specifically the 200 kDa neurofilaments and the myelin basic protein, in rats with brain ischemia-reperfusion injury treated with leuprolide acetate.

Methods:

The rats were divided into three groups: Sham, ischemia-reperfusion with saline solution, and ischemia-reperfusion treated with leuprolide acetate. Coronal brain slices were obtained and stained with hematoxylin-eosin. The histological analysis involved quantifying the number of neurons in the hippocampal regions CA1, CA3 and DG. The myelin basic protein and neurofilaments were quantified using western blot.

Results:

The number of neurons in CA1 and DG was significantly higher in the leuprolide acetate group compared to the untreated group. Additionally, the expression of neurofilament and myelin basic protein markers was significantly increased in rats treated with leuprolide acetate compared to the untreated rats.

Conclusions:

Leuprolide acetate promotes the recovery of hippocampal neurons in an acute brain ischemia-reperfusion injury model. These findings suggest that leuprolide acetate could be a potential therapeutic intervention for reversing damage in hippocampal ischemic lesions.

Keywords

Neuroregeneration myelin basic protein stroke neurofilaments

1 Introduction

Cerebral ischemia is a severe neurological disorder characterized by inadequate oxygen supply, leading to rapid deterioration of multiple brain regions. Among these regions, the hippocampus is particularly susceptible to damage following a stroke, as evidenced by studies conducted by dos Santos et al. (2020), Gad et al. (2020), Nozaki et al. (2001), and Pan et al. (2007). Survivors of cerebral ischemia often experience permanent disabilities, including motor, speech, learning, and memory impairments. Consequently, the quality of life for these patients is significantly diminished (Knecht et al., 2011).

Previous studies have reported the presence of gonadotropin-releasing hormone (GnRH) and its receptor in the rat hippocampus, specifically in the pyramidal neurons of the CA1 to CA4 regions and the granule cells of the dentate gyrus (DG) (Leblanc et al., 1988). Furthermore, it has been observed that GnRH in the hippocampus can induce changes in the density of dendritic spines (Prange-Kiel et al., 2008). Additionally, the GnRH analogue alarelin has been shown to attenuate apoptosis in rat hippocampal neurons following ischemia-reperfusion injury (Chu et al., 2010).

GnRH and its agonists have demonstrated neurotrophic properties. In rats with spinal cord injury, the administration of GnRH and leuprolide acetate (LA) has been shown to increase the expression of proteins such as neurofilaments (NFs) and myelin basic protein (MBP) in the injured spinal cord, leading to an improvement in locomotor activity (Calderón-Vallejo & Quintanar, 2012; Díaz Galindo et al., 2015; Guzmán-Soto et al., 2012). Furthermore, LA treatment has been found to decrease scar tissue formation in both gray and white matter, enhancing the preserved area in rats with spinal cord lesions (Díaz Galindo et al., 2015), and to improve functional recovery of nerve conduction velocity in rats with complete transection of the sciatic nerve (Hernández-Jasso et al., 2020).

Considering the previous background, we hypothesize that LA may have the potential to promote the healing of neurons damaged by ischemia-reperfusion injury. The objective of this study was to evaluate the histology of the hippocampus and assess the expression of neuronal recovery marker proteins, including the 200 kDa neurofilament (NF200) and MBP, in rats with brain ischemia-reperfusion injury treated with LA.

2 Materials and methods

2.1 Animals

Male Sprague-Dawley rats of 270 to 300 g were used for this study. The rats were sourced from the animal farm of the Autonomous University of Aguascalientes and were handled in compliance with the Institutional Normative Standards of the Ethics Committee for the Use of Animals in Teaching and Research of the Autonomous University of Aguascalientes. The animals were housed under controlled conditions, with a temperature of 22°C, a 12-hour light-dark cycle, and ad libitum access to Purina Chow food and water. Every effort was made to minimize animal suffering throughout the study.

2.2 Occlusion of the middle cerebral artery

The surgical procedure involved the occlusion of the middle cerebral artery by introducing an intraluminal suture thread through the left internal carotid artery. This was performed under anesthesia with Ketamine/Xylazine (70/10 mg/kg, intraperitoneally), following the protocol described by Longa et al. (1989). After a duration of 60 minutes, the suture was removed to allow reperfusion. Subsequently, all animals received an intradermal injection of 5 ml sterile saline solution for fluid recovery and Procaine benzylpenicillin (5,000 IU, intramuscularly).

To determine the infarcted area in the brain, a subgroup of five animals from each group was sacrificed via anesthesia overdose, and their brains were removed. Fresh coronal slices of tissue (each brain was cut into seven slices) were incubated in a 1% solution of 2,3,5-triphenyltetrazolium chloride (TTC) at 37°C for 20 minutes. The healthy area appeared stained in red, while the infarcted area remained white. The infarcted area (mm²) was calculated by measuring the area of the contralateral hemisphere and subtracting the healthy area of the ipsilateral hemisphere.

2.3 Leuprolide acetate treatment

The rats were divided into three groups: 1) sham surgery without ischemia-reperfusion (sham, n = 10), 2) ischemia-reperfusion treated with saline solution (IR+SS, n = 10), and 3) ischemia-reperfusion treated with LA (IR+LA, n = 10). The treatment protocol was based on the method described by Díaz-Galindo et al. (2015). Briefly, LA (Sigma, St. Louis, Cat: L0399) was administered at a dose of 10μg/kg (in a volume of 100μl per dose) via intramuscular injection one hour after reperfusion and repeated once every 24 hours for the next two consecutive days. Subsequently, the dose was administered every third day until day 28. The sham and IR+SS groups received intramuscular injections of 100μl physiological saline solution (0.9% NaCl) on the same days as the LA-treated rats. On day 28, all animals were euthanized under deep anesthesia with ketamine.

2.4 Neurological assessment

At 24 hours after the brain injury surgery, a neurological scale test was performed to assess the degree of damage according to the scale developed by Rousselet et al. (2012). The neurological signs observed in the rats were used to assign a score ranging from 0 to 5. Score 0 indicated a normal condition, score 1 represented slight circling behavior with or without inconsistent rotation when the rat was lifted by its tail, with less than 50% of attempts to turn towards the contralateral side. Score 2 indicated consistent smooth circling behavior, with more than 50% of attempts to turn towards the contralateral side. Score 3 represented consistent strong and immediate circling behavior, where the rat maintained a rotation position for more than 1-2 seconds, with its nose almost reaching its tail. Score 4 indicated severe rotation progressing to dragging, including loss of gait or righting reflex. Finally, score 5 represented a comatose or moribund state.

2.5 Histological analysis

The rats were sacrificed and perfused through the cardiac route with cold saline, followed by 10% neutral formalin. The brains were carefully removed and placed in the same fixative solution for 7 days at room temperature, shielded from light. Subsequently, the tissues were embedded in paraffin, and 9μm thick coronal sections were cut. The sections were obtained at the interaural distance of 5.4 mm in the bregma region of –3.60 mm, as described in the Paxinos and Watson atlas (Paxinos & Watson, 2005). Hematoxylin-eosin staining was performed on the sections. Photomicrographs were captured using a 40X objective lens with an LGE LM-X520 camera model. The camera settings included an ISO speed rating of 100, a focal length of 3.5 mm, and a variable exposure time ranging from 1/60 s to 1/30 s. Each image was captured at a resolution of 4161×3120 pixels and a resolution of 72 ppi. The images were originally saved in JPEG format. The total neuron count was determined using the method of neuron cell count with deep learning in highly dense hippocampus images, as reported by Vizcaíno et al. (2022). Two blinded experimenters performed the quantification of neurons.

2.6 Western blot analysis of NF200 and MBP

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10% SDS-PAGE) was performed using 30μg of protein from each hippocampal sample. The protein amount in each sample was determined by the Bradford method, and equal amounts of protein were used for comparison in each experiment. Following electrophoresis, the gels were electrotransferred onto polyvinylidene difluoride membranes, which were then incubated with primary antibodies against NF200 (1 : 500, Sigma, USA), MBP (1 : 1000, Invitrogen, USA), and polyclonal GAPDH (1 : 1000, Sigma, USA). Subsequently, the membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibodies. Chemiluminescence was used for membrane development, and the signals were visualized using Image Lab^TM (Bio-Rad). The relative density of the bands was analyzed using Quantity One®. Band densities were normalized using the signal intensity of GAPDH.

2.7 Statistical analysis

The data are presented as mean±SEM. One-way analysis of variance (ANOVA), followed by Tukey’s post hoc test and size effect were performed using GraphPad Prism version 9.00. Statistical significance was set at p < 0.05.

3 Results

One day after the brain injury surgery, a neurological scale test was performed to assess the degree of damage in all animals. The sham group did not show any signs of injury, while all animals in the two groups that underwent cerebral ischemia exhibited signs of brain injury. The mean score of the neurological lesion was 2.5, indicating intermediate severity.

To confirm the occurrence of brain injury due to middle cerebral artery occlusion, TTC staining was performed on the brains of the three groups. The global damage area in the brain was 18.4% for the IR+SS group and 11.9% for the IR+LA group. No infarct area was observed in the sham group.

Figure 1a displays representative photomicrographs of the CA1, CA3, and DG regions of the rat hippocampus in the three study groups. The first column shows images obtained with a 4X objective, while the next three columns show zoomed-in images taken with a 40X objective. In the photomicrographs of the CA1 and DG areas of the LA-treated animals, a greater number of neuronal cell bodies was observed compared to the other two groups. Quantitative analysis of the total number of neurons was performed in the CA1, CA3, and DG areas. The results revealed a significantly higher number of neurons in the CA1 and DG areas of the hippocampus in LA-treated rats compared to untreated animals (p < 0.05, Tukey’s test). However, the number of total neurons in the CA1 area was similar among the three study groups. In the DG region, the number of neurons was significantly higher in rats treated with LA compared to untreated animals (p < 0.001, Tukey’s test), and not different to the sham group (p > 0.05, Tukey’s test) (Fig. 1b).

Fig. 1

Effect of leuprolide acetate on the number of hippocampal neurons in CA1, CA3 and DG of rats with acute brain ischemia-reperfusion injury. a. Hippocampus photomicrographs of rats with surgery without occlusion (sham, n = 5), with brain ischemia-reperfusion treated with saline solution (IR + SS, n = 10), and with ischemia-reperfusion treated with leuprolide acetate (IR + LA, n = 7). The tissue was stained with hematoxylin-eosin. Scale bars in the hippocampus represent 500μm and in CA1, CA3 and DG 50μm. The frame indicates the area where the neuronal quantification was done. b. Total neurons count in the different hippocampal areas. Data are presented as mean±SEM. Analysis of variance (ANOVA) and Tukey’s comparison test were used. CA1: F = 4.618, dfN = 2, dfD = 22, *P = 0.02 (IR + LA vs IR + SS), size effec (d = 0.9). CA3: F = 1.829, dfN = 2, dfD = 10, P = 0.2105, size effect (d = 1.5). DG: F = 26.50, dfN = 2, dfD = 9, size effect (d = 1.1), **P = 0.0002 (IR + LA vs. IR + SS).

Western blot analysis demonstrated that NF200 protein expression was significantly higher in the hippocampus of LA-treated brain-injured rats compared to untreated rats (Fig. 2). Another protein analyzed using this technique was MBP, and the results also showed significantly higher expression of this protein in the hippocampus of injured rats that received LA treatment compared to untreated animals. The expression of NF200 and MBP was normalized using the constitutive GAPDH enzyme (Fig. 3).

Fig. 2

Western blot analysis of NF200 and GAPDH expression in the hippocampus of rats with acute brain ischemia-reperfusion injury. (A) Immunoblot representative of NF200 and GAPDH in the group with surgery without occlusion (sham), with ischemia-reperfusion injury treated with saline solution (IR + SS), and with ischemia-reperfusion injury treated with leuprolide acetate (IR + LA) for a 28-day period and (B) densitometric analysis. Values are expressed as optical density relative to GAPDH per 30μg of protein. F = 4.767, dfN = 2, dfD = 15, size effect (d = 0.9), *P = 0.0250.

Fig. 3

Immunoblot analysis of MBP and GAPDH expression in the hippocampus of rats with acute brain ischemia-reperfusion injury. (A) MBP immunoblot for the group with surgery without occlusion (sham), with ischemia-reperfusion injury treated with saline solution (IR + SS), and with ischemia-reperfusion injury treated with leuprolide acetate (IR + LA) for a 28-day period and (B) densitometric analysis. Values are expressed as optical density relative to GAPDH per 30μg of protein. F = 5.417, dfN = 2, dfD = 7, size effect (d = 1.5) *P = 0.0379.

4 Discussion

Our results demonstrate that the occlusion of the middle cerebral artery induces ischemia, leading to brain injury, particularly in the cerebral cortex and hippocampus. These findings are consistent with previous studies by Chu et al. (2010) and Longa et al. (1989).

In the histological analysis, we observed that the number of neurons in the CA1 and DG regions in animals with brain ischemia-reperfusion injury treated with LA was comparable to that in sham rats, and significantly higher than in the IR + SS group. Previous studies have demonstrated the heightened vulnerability of pyramidal neurons in CA1 to ischemic insults (Jennes et al., 1995). The number of CA3 neurons in the LA-treated group was similar to the untreated group. This fact may be attributed to a lower density of GnRH receptors in CA3 when compared to CA1 and DG (Albertson et al., 2008).

After cerebral ischemia, one possible cause of neuronal death in the hippocampus is apoptosis, a mechanism to which these neurons are particularly vulnerable (Kaplan & Miller, 2000). The presence of the GnRH receptor has been described in these neurons, particularly in the CA1, CA2, CA3, CA4, and DG areas (Jennes et al., 1995). It is plausible that the observed recovery in the number of neurons in the CA1 and DG regions in our experiments was due to a reduction in apoptosis resulting from LA treatment. Similar findings were reported in another study on rats with ischemia-reperfusion brain injury, wherein the administration of a GnRH analogue led to a decrease in apoptosis in the CA1 area of the hippocampus (Chu et al., 2010). Additionally, another possibility is the induction of neurogenesis in the CA1 and DG regions (Hainmueller & Bartos, 2020).

Myelin, composed of several structural proteins, is produced by oligodendrocytes. Among these proteins, MBP is highly abundant and has been extensively studied in various neurological disorders, including those with hippocampal involvement (Park et al., 2016). Previous studies using the middle cerebral artery occlusion (MCAO) model have demonstrated that myelin destruction initiates within hours or even on the first day after ischemia. Furthermore, oligodendrocytes have been shown to be highly susceptible to ischemic injury, resulting in their death and complicating the remyelination process for remaining axons (Dewar et al., 2003; McIver et al., 2010).

Our findings indicate that rats with brain ischemia-reperfusion injury treated with LA exhibited increased expression of MBP. To our knowledge, this is the first report demonstrating an elevation of MBP levels following treatment with a GnRH agonist after an ischemia-reperfusion brain injury. Another study also reported an upregulation of MBP expression following LA administration in the spinal cord of rats with experimental autoimmune encephalomyelitis, which aligns with our results (Guzmán-Soto et al., 2012). The underlying mechanism for this effect remains unknown; however, it is possible that LA treatment contributes to a reduction in oligodendrocyte apoptosis, thus promoting nerve fiber remyelination and ultimately leading to increased MBP expression.

NFs are crucial components of the neuronal cytoskeleton and play a vital role in maintaining axonal caliber, promoting neuronal growth, organizing neuronal structure, and facilitating plasticity. NFs have been identified in the rat hippocampus, specifically in the CA1-CA3 and DG regions (Lopez-Picon et al., 2003; Simonová et al., 2003).

Our results demonstrate a decrease in NF200 expression in the hippocampus following brain ischemia-reperfusion injury. Similar findings were observed in newborn rats exposed to hypoxia, where a significant reduction in NFs was observed in the CA1 and DG regions (Simonová et al., 2003). Conversely, animals treated with LA showed an increase in NF200 expression compared to those not treated with LA, similar to the sham group. This suggests that LA treatment may have induced axonal regeneration, leading to an upregulation of NFs, as observed in cultured spinal cord neurons incubated with GnRH (Quintanar et al., 2016), as well as in two different models of injury: spinal cord injury and experimental autoimmune encephalomyelitis (Calderón-Vallejo & Quintanar, 2012; Díaz Galindo et al., 2015; Guzmán-Soto et al., 2012).

5 Conclusion

The results of this study indicate that treatment with LA in rats with brain ischemia-reperfusion injury has a neurotrophic effect on CA1 and DG neurons, as well as on the expression of NF200 and MBP in the hippocampus. These findings suggest that LA may exert its effects in acute brain ischemia-reperfusion injury by reducing apoptotic processes and promoting neurogenesis and axonal regeneration. The present work highlights the potential of LA as a therapeutic agent for promoting neuronal recovery in hippocampal lesions.

Footnotes

Acknowledgments

We thank AQB. Sonia Sofía Cruz Muñoz and Dr. María Consolación Martínez Saldaña for the technical support. We thank CONACyT for the grant 214971.

Disclosure of conflicts of interest

On behalf of all authors, the corresponding author states that there are no conflicts of interest.

References

Albertson,

A.J.

, Navratil,

, Mignot,

, Dufourny,

, Cherrington,

, & Skinner,

D.C.

(2008) Immunoreactive GnRH type I receptors in the mouse and sheep brain. Journal of Chemical Neuroanatomy, 35(4), 326–333. https://doi.org/10.1016/j.jchemneu.2008.03.004

Calderón-Vallejo,

, & Quintanar,

J.L.

(2012) Gonadotropin-releasing hormone treatment improves locomotor activity, urinary function and neurofilament protein expression after spinal cord injury in ovariectomized rats. Neuroscience Letters, 515(2), 187–190. https://doi.org/10.1016/j.neulet.2012.03.052

Chu,

, Xu,

, & Huang,

(2010) GnRH analogue attenuated apoptosis of rat hippocampal neuron after ischemia-reperfusion injury. Journal of Molecular Histology, 41(6), 387–393. https://doi.org/10.1007/s10735-010-9300-8

Dewar,

, Underhill,

S.M.

, & Goldberg,

M.P.

(2003) Oligodendrocytes and ischemic brain injury. Journal of Cerebral Blood Flow and Metabolism: Official Journal of the International Society of Cerebral Blood Flow and Metabolism, 23(3), 263–274. https://doi.org/10.1097/01.WCB.0000053472.41007.F9

Díaz Galindo,

, Gómez-Gonzáez,

, Salinas,

, Calderón-Vallejo,

, Hernández-Jasso,

, Bautista,

, & Quintanar,

J.L.

(2015) Leuprolide acetate induces structural and functional recovery of injured spinal cord in rats. Neural Regeneration Research, 10(11), 1819–1824. https://doi.org/10.4103/1673-5374.170311

dos Santos,

I.R.C.

, Dias,

M.N.C.

, & Gomes-Leal,

(2020) Microglial activation and adult neurogenesis after brain stroke. Neural Regeneration Research, 16(3), 456–459. https://doi.org/10.4103/1673-5374.291383

Gad,

S.N.

, Nofal,

, Raafat,

E.M.

, & Ahmed,

A.A.E.

(2020) Lixisenatide Reduced Damage in Hippocampus CA Neurons in a Rat Model of Cerebral Ischemia-Reperfusion Possibly Via the ERK/P38 Signaling Pathway. Journal of Molecular Neuroscience, 70(7), 1026–1037. https://doi.org/10.1007/s12031-020-01497-9

Guzmán-Soto,

, Salinas,

, Hernández-Jasso,

, & Quintanar,

J.L.

(2012) Leuprolide acetate, a GnRH agonist, improves experimental autoimmune encephalomyelitis: A possible therapy for multiple sclerosis. Neurochemical Research, 37(10), 2190–2197. https://doi.org/10.1007/s11064-012-0842-x.

Hainmueller,

, & Bartos,

(2020) Dentate gyrus circuits for encoding, retrieval and discrimination of episodic memories. Nature Reviews. Neuroscience, 21(3), 153–168. https://doi.org/10.1038/s41583-019-0260-z

10.

Hernández-Jasso,

, Domínguez-Del-Toro,

, Delgado-García,

J.M.

, & Quintanar,

J.L.

(2020) Recovery of sciatic nerve with complete transection in rats treated with leuprolide acetate: A gonadotropin-releasing hormone agonist. Neuroscience Letters, 739, 135439. https://doi.org/10.1016/j.neulet.2020.135439

11.

Jennes,

, Brame,

, Centers,

, Janovick,

J.A.

, & Conn,

P.M.

(1995) Regulation of hippocampal gonadotropin releasing hormone (GnRH) receptor mRNA and GnRH-stimulated inositol phosphate production by gonadal steroid hormones. Brain Research. Molecular Brain Research, 33(1), 104–110. https://doi.org/10.1016/0169-328x(95)00113-7

12.

Kaplan,

D.R.

, & Miller,

F.D.

(2000) Neurotrophin signal transduction in the nervous system. Current Opinion in Neurobiology, 10(3), 381–391. https://doi.org/10.1016/s0959-4388(00)00092-1

13.

Knecht,

, Hesse,

, & Oster,

(2011) Rehabilitation After Stroke. Ärzteblatt Int, 108, 600–606. https://doi.org/10.3238/arztebl.2011.0600

14.

Leblanc,

, Crumeyrolle,

, Latouche,

, Jordan,

, Fillion,

, L’Heritier,

, Kordon,

, Dussaillant,

, Rostène,

, & Haour,

(1988) Characterization and distribution of receptors for gonadotropin-releasing hormone in the rat hippocampus. Neuroendocrinology, 48(5), 482–488. https://doi.org/10.1159/000125053

15.

Longa,

E.Z.

, Weinstein,

P.R.

, Carlson,

, & Cummins,

(1989) Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke, 20(1), 84–91. https://doi.org/10.1161/01.str.20.1.84

16.

Lopez-Picon,

F.R.

, Uusi-Oukari,

, & Holopainen,

I.E.

(2003) Differential expression and localization of the phosphorylated and nonphosphorylated neurofilaments during the early postnatal development of rat hippocampus. Hippocampus, 13(7), 767–779. https://doi.org/10.1002/hipo.10122

17.

McIver,

S.R.

, Muccigrosso,

, Gonzales,

E.R.

, Lee,

J.M.

, Roberts,

M.S.

, Sands,

M.S.

, & Goldberg,

M.P.

(2010) Oligodendrocyte degeneration and recovery after focal cerebral ischemia. Neuroscience, 169(3), 1364–1375. https://doi.org/10.1016/j.neuroscience.2010.04.070

18.

Nozaki,

, Nishimura,

, & Hashimoto,

(2001) Mitogen-activated protein kinases and cerebral ischemia. Molecular Neurobiology, 23(1), 1–19. https://doi.org/10.1385/MN:23:1:01

19.

Pan,

, Konstas,

A.-A.

, Bateman,

, Ortolano,

G.A.

, & Pile-Spellman,

(2007) Reperfusion injury following cerebral ischemia: Pathophysiology, MR imaging, and potential therapies. Neuroradiology, 49(2), 93–102. https://doi.org/10.1007/s00234-006-0183-z

20.

Park,

J.H.

, Choi,

H.Y.

, Cho,

J.-H.

, Kim,

I.H.

, Lee,

T.-K.

, Lee,

J.-C.

, Won,

M.-H.

, Chen,

B.H.

, Shin,

B.-N.

, Ahn,

J.H.

, Tae,

H.-J.

, Choi,

J.H.

, Chung,

J.-Y.

, Lee,

C.-H.

, Cho,

J.H.

, Kang,

I.J.

, & Kim,

J.-D.

(2016) Effects of Chronic Scopolamine Treatment on Cognitive Impairments and Myelin Basic Protein Expression in the Mouse Hippocampus. Journal of Molecular Neuroscience: MN, 59(4), 579–589. https://doi.org/10.1007/s12031-016-0780-1.

21.

Paxinos,

Watson,

Ch.

(2005) The Rat Brain in stereotaxic coordenates (5ta ed.). Elsevier.

22.

Prange-Kiel,

, Jarry,

, Schoen,

, Kohlmann,

, Lohse,

, Zhou,

, & Rune,

G.M.

(2008) Gonadotropin-releasing hormone regulates spine density via its regulatory role in hippocampal estrogen synthesis. The Journal of Cell Biology, 180(2), 417–426. https://doi.org/10.1083/jcb.200707043

23.

Quintanar,

J.L.

, Calderón-Vallejo,

, & Hernández-Jasso,

(2016) Effects of GnRH on Neurite Outgrowth, Neurofilament and Spinophilin Proteins Expression in Cultured Spinal Cord Neurons of Rat Embryos. Neurochemical Research, 41(10), 2693–2698. https://doi.org/10.1007/s11064-016-1983-0.

24.

Rousselet,

, Kriz,

, & Seidah,

N.G.

(2012) Mouse model of intraluminal MCAO: Cerebral infarct evaluation by cresyl violet staining. Journal of Visualized Experiments: JoVE, 69, 4038. https://doi.org/10.3791/4038

25.

Simonová,

, Sterbová,

, Brozek,

, Komárek,

, & Syková,

(2003) Postnatal hypobaric hypoxia in rats impairs water maze learning and the morphology of neurones and macroglia in cortex and hippocampus. Behavioural Brain Research, 141(2), 195–205. https://doi.org/10.1016/s0166-4328(02)00366-2

26.

Vizcaíno,

, Sánchez-Cruz,

, Sossa,

, & Quintanar,

J.L.

(2022) Neuron cell count with deep learning in highly dense hippocampus images. Expert Systems with Applications, 208, 118090. https://doi.org/10.1016/j.eswa.2022.118090