Key words : Cell transplant, Neurological disorders, Stroke

Introduction

Stroke exacts a heavy toll on death and disability worldwide. Every year, thousands of people have brain injuries and strokes. Unfortunately, the damaged brain has limited ability,¹ and no effective neuroprotective treatment has been found.² Transplantation of stem cells can improve the deficits of brain ischemia. Transplantation of neural stem cells, which are extracted from embryonic cells, has been shown to improve deficits in an animal model of brain ischemia.³ Sertoli cells play a role by secreting various factors that actively inhibit the immune response. These cells, derived from testes, have been used to create an out-of-testicular immune system to facilitate cell transplant protocols for neuronal diseases. In addition to the secretion of immune inhibitor factors, Sertoli cells also release growth factors that appear to increase the survival of transplanted cells. Therefore, the cotransplant of Sertoli cells with grafted cells can be beneficial.⁴

Rapamycin, a potent agent for suppressing the immune system, belongs to the class of macro-silicon suppressant drugs that are activated only when they are restricted to immunophilins.⁵ Rapamycin is also a macrolide antibiotic that can reduce various types of nerve damage by activating autophagy. The mechanistic target of rapamycin (mTOR) pathway is a crucial intracellular regulator of the immune system.⁶ In addition, the mTOR signaling pathway regulates different processes, such as immune responses and cell growth, and has a critical role in the nervous system. Rapamycin has a protective role in the central nervous system by inhibiting the mTOR signaling pathway, resulting in autophagy.⁷ In an animal model of intracerebral hemorrhage, inhibition of the mTOR pathway modulated the immune response and also improved neurobehavioral deficits in the animals.⁷ Conversely, mTOR pathway inhibition has also been shown to ameliorate neural damage and improve memory function and synaptic plasticity in Alzheimer disease, as well as assist in long-term memory in the amygdala.⁸

In this study, our aim was to evaluate the possible effect of rapamycin injection plus Sertoli cell transplant in a rat model of neurological deficits.

Materials and Methods

Animal
Adult rats weighing between 250 and 300 g were obtained from the Pasteur Institute Center and kept in appropriate numbers in separate cages under standard conditions (temperature of 22 ± 2 °C and 12:12 hour light-dark cycle).

Experimental protocols
The 75 adult male rats were divided into 5 main groups: sham group, control group, and 3 treatment groups (rapamycin administration, Sertoli cell transplant, and Sertoli cell transplant plus rapamycin administration). Middle cerebral artery occlusion (MCAO) surgery was performed in the control group. The sham group only received surgical stress without ischemia. The rapamycin-only group received intraperitoneally injected rapamycin at 300 mg/kg at 1 hour before the MCAO surgery, the Sertoli cell transplant group received injection of 500?000 Sertoli cells into the right striatum of the brain by stereotaxic with MCAO surgery after 5 days, and the combination group of both rapamycin and Sertoli cells received treatment as described for the single-treatment groups. Each group contained 15 rats.

Ethical statement
Our laboratory animal protocols complied with the National Institutes of Health guidelines for the humane use and care of animals and has been affirmed by our institutional ethics committee.

Preparation of Sertoli cells
Testicles were removed from killed rats, cut into small pieces, and then transferred into Falcon tubes containing conditioning media and antibiotics. Tissues were then incubated with 25% trypsin at 37 °C for 15 minutes to separate seminiferous tubules. After aspiration of trypsin, 1% collagenase solution was added, with incubation continued for another 15 minutes. Fetal bovine serum was added, and samples were centrifuged. The cell suspension was transferred into culture flasks containing Dulbecco’s modified Eagle medium/F12 medium with 10% fetal bovine serum and antibiotics.

Immunocytochemistry of Sertoli cells
After Sertoli cells were checked for density, they were washed with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde solution. Next, culture plates were incubated with 0.3% Triton X-100 solution (Sigma Aldrich) and then incubated overnight with primary antibody (anti-GATA4). Cells were then washed again with PBS and incubated with the second antibody (goat conjugated FITC anti-mouse antibody). After a repeat washing and staining of cores with Hoechst, samples were examined under a fluorescent microscope.

Stereotaxic surgery and injection of Sertoli cells
Sertoli cells were first incubated with Hoechst for several minutes; after cells reached the appropriate density, they were separated with trypsin and centrifuged and counted with Trypan blue. Rats were first anesthetized, and then their heads were fixed in a stereotaxic device and skull surfaces were cleaned. After the distance between the Lambda and Bregma was found, 500?000 cells were injected into the right striatum with the use of a Hamilton microsyringe, according to following coordinates of the rat brain atlas from Bregma: anterior-posterior = +0.5 mm, medial-lateral = ±2.6 mm, and dorsal ventral = -5 mm.

Pharmaceutical compound
Rapamycin was obtained from Cayman Pharmaceutical Company. A rapamycin dose of 300 mg/kg was dissolved in dimethyl sulfoxide and injected intraperitoneally 1 hour before induction of MCAO. Rapamycin has the ability to induce apoptosis in cancer cells and suppress mTOR signaling in cancer treatment. It is also an immunosuppressant and widely used in scientific research.⁹

Focal cerebral ischemia
After rats were anesthetized with 400 mg/kg chloral hydrate intraperitoneally (Merck), they underwent surgery according to the method from Longa and colleagues.¹⁰ A 3-0 nylon suture, 4 cm long, was passed through the common carotid. As a result of insertion of the suture and blockage of arterial blood flow, blood flow did not to reach the middle cerebral artery. After placement for 60 minutes, we slowly removed the suture.

Neurobehavioral evaluation
After placement in intensive care for 24 hours, rats underwent neurological tests. Findings were grouped into 5 scales: motor function, sensory function, raise the tail, beam test, and reflex activity The highest score was 18.¹¹

Measurement of cerebral ischemic volume
After rats were killed with a high dose of anesthetic drugs, rat brains were removed and placed in cold saline for 5 minutes. Brains were then placed in a matrix and cut to a thickness of 2 mm. Slices were placed in a bath for 15 minutes at 37 °C for staining with 2% triphenyl tetrazolium chloride solution.¹² White color and red color indicated ischemic and healthy tissue, respectively. Infarct volume of each section was analyzed separately using the following formula: corrected volume of damaged area = left hemisphere volume – (right hemisphere volume – damaged area volume).

Measurement of blood-brain barrier permeability
The strength of the blood-brain barrier was assessed by the departure of Evans blue. Thirty minutes after induction of ischemia, 2% Evans blue solution (in the amount of 4 mL/kg) was injected through the tail vein. Twenty-four hours later, with the use of 250 mL of saline, the arteries were cleared of Evans blue. After the brain was removed, parts of the cortex, piriform cortex-amygdala, and striatum were separated and homogenized with PBS. After homogenization with 2.5 cm³ of 60% trichloroacetic acid, tissue samples were placed at 4 °C for 30 minutes and then centrifuged at 1000 revolutions/min. Light absorption of the brain solution was then measured at 610 nm.¹³

Measurement brain water level
Wet weights of the brain, including the cortex, piriform cortex-amygdala, and striatum, were calculated. After incubation at 120 °C for 24 hours, the dry weight was also measured. Brain water content was then calculated based on the following formula13: ([wet weight – dry weight]/wet weight) × 100.

Statistical analyses
Neurological evaluation was calculated using the nonparametric Kruskal-Wallis test. The volume of tissue damage, brain edema, and blood brain-barrier permeability were analyzed using one-way analysis of variance, followed by the Bonferroni post hoc test. Significance levels were set at P < .05.

Results

Confirming presence of derived cells from testes
The immunocytochemistry results of Sertoli cells against anti-GATA4 antibody showed that the cultured cells expressed the GATA4 marker; GATA4 expressed in Sertoli cells had stained the cells in green. Hoechst staining demonstrated nuclei of cells, with blue nuclei representing those in Sertoli cells (Figure 1A). Sertoli cells were injected with DiI stain to show viability of injected cells in the striatum after 1 week. We also used DiI labeling to tag transplanted Sertoli cells (Figure 1B).

Effect of rapamycin injection and Sertoli cell transplant on neurologic severity scores
Treatment of rats with Sertoli cells and rapamycin decreased brain evaluation scores, with score of 11.5 (range, 8-12) in rats that received Sertoli cells only, score of 7 (range, 5-11) in rats that received rapamycin only, and score of 6 (range, 3-12) in rats that received both rapamycin injection and Sertoli cell transplant (Table 1). The control group had a score of 14.5 (range, 13-15). Figure 2 shows results of neurological defect evaluations after 24 hours in sham (no ischemic injury), rapamycin only, Sertoli cell only, and the combined rapamycin plus Sertoli cell group and compared with the control group.

Effect of rapamycin injection and Sertoli cell transplant on infarct volume
Total volume of strokes in animals was determined in the rapamycin group (P = .03), the Sertoli cell group (P = .001), and rapamycin plus Sertoli cell group (P = .007) versus the control group (Figure 3B). Cortical infarctions were reduced in the rapamycin group (P = .004), the Sertoli cell group (P = .02), and the rapamycin injection plus Sertoli cell group (P = .02) (Figure 3C). The piriform cortex-amygdala infarct volume in the rapamycin (P = .02), Sertoli cell transplant (P = .04), and rapamycin injection plus Sertoli cell transplant (P = .006) groups was also diminished compared with the control group (Figure 3D). Infarct volume of the striatum was also reduced in the rapamycin (P = .04), the Sertoli cell (P = .03), and rapamycin plus Sertoli cell treatment groups (P = .005) (Figure 3E).

Effect of rapamycin injection and Sertoli cell transplant on blood-brain barrier permeability
Compared with results shown in the control group, the MCAO procedure caused damage in the right hemisphere compared with the left hemisphere (P < .05). No significant differences were shown in the left hemispheres of the treatment groups versus the control group, illustrating that the blood-brain barrier was not impaired in the left hemisphere (P > .05). As shown in the right cortex, rapamycin (P = .04), Sertoli cell (P = .01), and rapamycin plus and Sertoli cell treatment (P = .02) were effective in reducing blood-brain barrier permeability after ischemia induction (Figure 4A). As shown in Figure 4B, decreased permeability was also shown in the piriform cortex-amygdala of all 3 treatment groups (P = .04, P = .001, and P = .009, respectively) versus the control group. The striatum also showed decreased permeability in all 3 treatment groups (P = .001, P = .005, and P = .01, respectively) (Figure 4C). The control group was also compared with the sham group (data not shown).

Effect of rapamycin injection and Sertoli cells transplant on brain water content
Cerebral edema in the 3 brain regions showed a decreasing trend compared with the control group. As shown in Figure 5A, decreasing edema was shown in the cortex of the rapamycin (P = .01), Sertoli cell (P = .03), and rapamycin plus Sertoli cell groups (P = .02). Figure 5B shows that brain edema in the piriform cortex-amygdala was also reduced in the rapamycin (P = .002), Sertoli cell (P = .002), and rapamycin plus Sertoli cell treatment groups (P = .001). Similarly, brain edema was reduced in the striatum of all 3 treatment groups (P = .002, P = .006, and P = .001, respectively) (Figure 5C). The control group was also compared with the sham group (data not shown).

Discussion

Recent evidence has suggested that rapamycin is neuroprotective, not only via neuronal autophagy but also through its broader effects on other cells of the neurovascular unit.¹⁴ Rapamycin, an antibiotic produced from Streptomyces hygroscopicus and a potent immunosuppressor, has been recently heralded for its anticancer properties. Rapamycin and its derivatives (rapalogs), such as the ester of rapamycin, CCI-779, are now showing significant activity against a variety of cancers.¹⁵ Various cell lines can exhibit differences in their sensitivity to rapamycin under similar growth conditions. The mTOR pathway is highly conserved from yeast to human and promotes cell growth in response to nutrient availability. Therefore, mTOR inhibition through rapamycin treatment mimics a nutrient starvation phenotype induced by inhibition of protein synthesis, acquisition of thermotolerance, autophagy, and glycogen accumulation.¹⁶ The mTOR kinase, a master regulator that is evolutionarily conserved among yeasts, plants, animals, and humans, integrates nutrient and energy signaling to promote cell proliferation and growth.¹⁷ It is involved in the function of multiple cells; in a global sense, many of these functions are related to overall cell growth, survival, and homeostasis. Therefore, mTOR inhibition may be used to treat a variety of diseases.¹⁸

In addition to its effects on cell growth and promotion, the mTOR pathway is involved in the regulation of a number of neurological functions, such as neurotransmitter receptors, ion channel expression, and synaptic plasticity. Rapamycin, which can protect neurons by autophagy¹⁹ and is an inhibitor of mTOR, may be an effective treatment. Rapamycin has been demonstrated to exhibit neuroprotective functions via the activation of autophagy in a cerebral ischemia model.²⁰ However, the involvement of autophagy in this process and its contribution to the protection of mitochondrial function remain unknown. In the present study, we explored the characteristics of autophagy after cerebral ischemia and the effect of rapamycin on mitochondrial function.

Rapamycin can enhance autophagy, as evidenced by increases in LC3-II and Beclin-1 expression in
the mitochondria and p62 translocation to the mitochondria.²¹ Rapamycin has been shown to protect the blood-brain barrier, leading to reduced brain edema after focal cerebral ischemia reperfusion, in a rodent model via multiple mechanisms including reduced endothelial cell death via induction of autophagy, improved tight junction expression, inhibition of matrix metalloproteinase 9 (MMP9), and reduced aquaporin 4 (AQP4) expression.¹⁴ In the present study, we found that rapamycin treatment attenuated mitochondrial dysfunction following cerebral ischemia, which is linked to enhanced autophagy.

Autophagy during mTOR inhibition results in neuroprotection. The blockage of mTOR signaling with the induction of autophagy can lead to neural tissue protection and functional improvements, as shown in a spinal cord injury model.²² Evidence has also shown that autophagy can contribute to cell death in many pathophysiological conditions, such as stroke. It has therefore been interpreted as a protective response to stress.²³ Rapamycin may also improve neuronal survival after oxygen deprivation. In an in vitro model, rapamycin enhanced neural survival after oxygen deprivation, which probably occurred throughout autophagy.²⁴ Administration of rapamycin 1 hour after MCAO surgery has also resulted in neuroprotective effects by improving antioxidant systems and decreasing inflammatory substances such as nitric oxide.²⁵ In an animal model of brain ischemia, injection of rapamycin into the hippocampus 24 hours before MCAO reduced neurologic deficit scores, infarct volume, and brain edema.²⁶ Rapamycin also preserved the blood-brain barrier structure and suppressed brain edema following brain ischemia via MMP9 and AQP4 inhibition.²⁷ After ischemia, MMP9 function in the brain may increase, which can disrupt the permeability of the blood-brain barrier in animal and human models.²⁸

Cell transplant therapy has received a great deal of attention for its role in stroke injuries. Cell transplant exerts its effects in healing this type of brain injury through a variety of mechanisms.²⁹ One mechanism is cell replacement. Another effective mechanism with this type of treatment is that the transplanted cells either secrete trophic factors directly or force the recipient’s nerve cells to secrete these factors.³⁰

The therapeutic effects of Sertoli cell transplant have been shown in vivo. In an animal model of autoimmune diseases, administration of Sertoli cells resulted in immunosuppression and ameliorated deficits of neurological diseases, such as Parkinson and Huntington.^31,32 The findings reported on glial cell-derived neurotrophic factor (GDNF) as a new neurotrophic factor for the production of peripheral neurons. Studies have shown that transplanting nerve cells into the brains of stroke patients increased ischemic tolerance and increased the expression of antioxidant enzymes, growth factors, GDNF, and vascular endothelial growth factor (VEGF).^33,34 Injection of Sertoli cells with other cells into the rat brain can cause cell survival. The Sertoli cell provides a suitable environment for the development of germ cells that have high antigenic properties.³⁵

Vascular endothelial growth factor plays a key role in the formation of physiological vessels and pathological angiogenesis, such as tumor growth and ischemic diseases.³⁶ Hypoxia is a strong inducer of VEGF in vitro. Its induction occurs in the brain as well as in the kidneys, testes, lungs, heart, and liver. A specific subset of cells in specific organs, such as glial cells and neurons in the brain, nephrons, and Sertoli cells in the testis, can respond to hypoxia by increasing VEGF expression.³⁷ Finally, VEGF, by increasing vascular density in the affected area, to some extent compensates for the loss of blood supply and plays an important role in revitalizing the area and reducing stroke volume.³⁸

Sertoli cells and surrounding tissues have enzymatic activities. They also increase sperm function, stimulation, and development by secreting trophic and regulatory factors.^39,40 The protein kinase signaling pathway, which is regulated by follicle-stimulating hormone, has an essential role in Sertoli cells in neonatal rats. Follicle-stimulating hormone enhances AKT signaling through insulin-like growth factor 1, which has been shown to interfere with cell survival and proliferation.^34,41

Explorations into the role of mTOR in neuro-vascular and neuroimmune regulation are at their infancy. An increased understanding of how this pathway contributes to pathology will be key for developing novel therapies. Further investigations are needed into the precise molecular mechanisms of rapamycin, the most efficacious dosing, and optimal timing in relation to ischemic stroke.

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