|Year : 2018 | Volume
| Issue : 1 | Page : 74-84
The possible therapeutic effect of mesenchymal stem cells on experimentally induced brain hypoxic-ischemic encephalopathy in neonatal rats
Nesrine Ebrahim1, Mohamed Y Salem1, Dina Sabry2, Ashraf Shamaa3
1 Department of Histology and Cell Biology, Faculty of Medicine, Banha University, Banha, Egypt
2 Department of Medical Biochemistry, Faculty of Medicine, Cairo University, Cairo, Egypt
3 Department of Surgical and Radiology, Faculty of Veterinary Medicine, Cairo University, Cairo, Egypt
|Date of Submission||18-Jan-2017|
|Date of Acceptance||21-Feb-2017|
|Date of Web Publication||28-Feb-2018|
Mohamed Y Salem
Farid Nada Street, Department of Histology and Cell Biology, Faculty of Medicine, Benha University, Benha - 13518
Source of Support: None, Conflict of Interest: None
Background Hypoxic-ischemic encephalopathy (HIE) in neonates is a serious disorder, and till now, no curable treatment is available.
Aim The present study was conducted to evaluate the therapeutic effect of bone marrow-derived mesenchymal stem cells (BM-MSCs) in attenuating the experimentally induced brain HIE in neonatal rats.
Materials and methods Totally, 45 healthy neonatal rats (7 days old) were divided into three groups: group I was the control group. In group II (encephalopathy group), rats were exposed to surgical induction of hypoxic-ischemic (HI) brain injury and subdivided into two subgroups: group IIa rats were killed 3 days later to evaluate the histopathological changes and group IIb rats were killed after 28 days for recovery. In group III (encephalopathy+stem cell group), rats were exposed to surgical induction of HI brain injury and then underwent infusion with BMSCs through tail vein. Two behavior tests (the righting reflex and the geotaxis reflex) were performed 3, 7, 14, and 28 days after induction of HI brain injury. Cerebral cortex tissue was processed for histological and immunohistochemical (nestin, glial fibrillar acid protein, and neurofilaments) studies and for real-time PCR quantitative expression of vascular endothelial growth factor-receptor 2 and endothelial nitric oxide synthase genes.
Results Group IIb (recovery) showed nearly the same picture as group IIa. Group III showed improvement in all parameters (neural function tests, histopathological studies, and genes expression) compared with group II.
Conclusion BM-MSCs attenuated the HIE in rats, but compared with the normal control, this effect was still away.
Keywords: cerebral cortex, neonatal hypoxic ischemia, stem cells
|How to cite this article:|
Ebrahim N, Salem MY, Sabry D, Shamaa A. The possible therapeutic effect of mesenchymal stem cells on experimentally induced brain hypoxic-ischemic encephalopathy in neonatal rats. Benha Med J 2018;35:74-84
|How to cite this URL:|
Ebrahim N, Salem MY, Sabry D, Shamaa A. The possible therapeutic effect of mesenchymal stem cells on experimentally induced brain hypoxic-ischemic encephalopathy in neonatal rats. Benha Med J [serial online] 2018 [cited 2018 May 23];35:74-84. Available from: http://www.bmfj.eg.net/text.asp?2018/35/1/74/226422
| Introduction|| |
Encephalopathy is a nonspecific term that refers to diffuse dysfunction within the brain that causes disturbances in function and mental status. Encephalopathy is usually accompanied with structural changes within the brain and metabolic disturbances. Traumatic brain injury, cerebrovascular accident, toxic agents, systemic disease, and genetic susceptibility are the most common causes of encephalopathy .
Hypoxic-ischemic encephalopathy (HIE) is a brain damage secondary to partial or total hypoxia that reduces blood flow to the brain of neonates. Serious and permanent neural defects such as cerebral palsy, epilepsy, memory deficiency, hypothermia, and even death may happen in severe cases ,. Neonates with similar degrees of hypoxic ischemia (HI) may differ in the extent of brain injury, ranging from none to near-total brain injury . HIE is a huge problem all over the world, as 10–60% of all affected infants die, and at least 25% of survivors have long-term neurologic complications. Its incidence is high (26 per 1000 live births) in underdeveloped countries ,.
Neurologic dysfunction early after HIE is the best indicator for neurodevelopmental outcome or death. Amplitude-integrated electroencephalography and MRIs have become widely available and have attained an important role in the prognosis of mild to moderate HIE . There is no standard therapy for HIE; however, many potential therapies that may prevent injury progression and enhance repair of brain injury are under investigation .
Mesenchymal stem cells (MSCs) have the capacity of renewal and differentiation into a variety of tissue lineages such as osteoblasts, chondrocytes, and adipocytes . MSCs can be derived from various tissues such as bone marrow, adipose tissue, and umbilical cord . MSCs have low immunogenicity, antiapoptotic, antifibrotic, and anti-inflammatory effects through the secretion of bioactive trophic factors that make them suitable for cellular therapy and regenerative medicine . MSC therapies provide a good chance for treating diseases of the brain, which have limited regenerative capacity .
The present study was conducted as an effort to evaluate the therapeutic effect of bone marrow-derived mesenchymal stem cells (BM-MSCs) in attenuating the experimentally induced brain HIE in neonatal rats.
| Materials and methods|| |
Totally, 45 healthy neonatal (7 days old) male albino rats weighing approximately 20 g were bred and maintained in an air conditioned animal house with specific pathogen-free conditions. Rats were subjected to a normal light/dark cycle and allowed unlimited access to chow and water. All animal procedures were performed according to the approved protocols of the Animal Committee of the Faculty of Medicine, Cairo University, and in accordance with the recommendations for the proper care and use of laboratory animals.
Rats were divided into three groups. Group I (control group) had 15 rats that were subdivided equally into three subgroups: group Ia − negative control rats; group Ib − sham operated control, where rats were exposed to the surgical procedures without bilateral cephalic arteries ligation, and received no pharmacological treatment; and group Ic − as sham operated control and rats were injected once with 0.5 ml of PBS intravenously through tail vein. Group II (encephalopathy group) had 20 rats that were exposed to surgical induction of HI brain injury; 10 rats were killed 3 days later to evaluate the histopathological changes (group IIa) and other 10 rats were killed after 28 days for recovery (group IIb). Group III (encephalopathy+stem cell group) had 10 rats that were exposed to surgical induction of HI brain injury and then underwent infusion with 107 MSCs/rat intravenously through tail vein ; rats were killed 28 days after induction of HI brain injury.
Induction of hypoxic-ischemic brain injury
HI brain injury was made by bilateral cephalic arteries ligation after ether anesthesia. Before the surgery, the animals were anesthetized by intraperitoneal injection of 4% chloral hydrate (0.5 ml/100 mg body weight). A 0.5-cm incision was made at the ventral side of the neck through which the cephalic arteries were isolated and ligated. The incision was then closed with stitches. Afterward, the animals were placed in an incubation chamber (37°C) for 10 min until their body temperature and behavior return to normal, and they were allowed to rest for another hour before being placed in a container aerated with 8% O2 and 92% N2 for 1.5 h at 37°C .
Preparation of bone marrow-derived mesenchymal stem cells
Rats’ BM-MSCs were prepared in the Biochemistry Department, Faculty of Medicine, Cairo University. Bone marrow cells were flushed from tibia and fibula of rat bones with PBS containing 2-ml EDTA. Over 15-ml Ficoll–Paque (Gibco Invitrogen, Grand Island, New York, USA), 35 ml of the diluted sample was carefully layered, and it was centrifuged for 35 min at 400g, and the upper layer was aspirated leaving undisturbed mononuclear cell layer at the interphase. This mononuclear cell layer was aspirated, washed twice in PBS containing 2-ml EDTA and centrifuged for 10 min at 200g at 10°C. The cell pellet was resuspended in a final volume of 300 μl of buffer. Isolated MSCs were cultured on 25-ml culture flasks in minimal essential medium supplemented with 15% fetal bovine serum and incubated for 2 h at 37°C and 5% CO2. Adherent MSCs were cultured in minimal essential medium supplemented with 30% fetal bovine serum, 0.5% penicillin, and streptomycin at 37°C in 5% CO2 in air . MSCs culture was done according to Yamazoe et al. . All cultures were examined using an inverted microscope; Leica DM IL LED with camera Leica DFC295 (Leica Microsystems CMS GmbH; Ernst-Leitz-Straße 17–37, Wetzlar, Germany).
Labeling stem cells with green fluorescent protein
MSCs labeled with green fluorescent protein  were observed in cerebral cortex cryosections using fluorescence microscope (Leica Microsystems CMS GmbH).
Neural function evaluation
Two behavior tests, the righting reflex and the geotactic reflex , were performed 3, 7, 14, and 28 days after induction of HI brain injury. The time (in seconds) needed to finish the righting reflex and the geotactic reflex was recorded.
Histological and immunohistochemical studies
The fasted rats were anesthetized by ether and killed by means of cervical decapitation. Cerebral cortex tissue was excised. Sections from the posterior parietal lobe of the cerebral cortex were fixed in 10% buffered formalin saline and processed for paraffin sections of 4–6 μm thickness, mounted on glass slides for hematoxylin and eosin staining . Other sections were mounted on positive-charged slides for immunohistochemical staining  using antinestin antibodies at 1 : 200 dilution, monoclonal antiglial fibrillary acidic protein (GFAP) antibody at 1 : 400 dilution, and antineurofilament (NF) 200 antibody at 1 : 80 dilution (Sigma-Aldrich, St. Louis, Missouri, USA). Antigen retrieval was performed in all cases by steam heating the slides in a 1 mmol/l solution of EDTA (pH 8.0) for 30 min. After blocking of endogenous biotin, staining was performed using an automated immunostainer followed by using a streptavidin–biotin detection system (Dako, Glostrup, Denmark). Sections were counterstained with hematoxylin. As a negative control, the primary antibody was replaced with PBS.
The mean area percentage of nestin, GFAP, and NFs immunoexpression were quantified five images from five nonoverlapping fields of each rat of each group using Image-Pro Plus Program version 6.0 (Media Cybernetics Inc., Bethesda, Maryland, USA).
Real-time quantitative PCR
Tissue of all studied groups was homogenized, and total RNA was isolated with RNeasy Mini Kit (Qiagen, Hilden, Germany) and further analyzed for quantity and quality with a dual-beam spectrophotometer (Beckman Coulter, Fullerton, California, USA). For quantitative expression of vascular endothelial growth factor-receptor 2 (VEGF-R2) and endothelial nitric oxide synthase (eNOS) genes, the following procedure was assessed: 200 ng of the total RNA from each sample was used for cDNA synthesis by reverse transcription using high-capacity cDNA Reverse Transcriptase Kit (Applied Biosystems Inc., Foster City, California, USA). The cDNA was subsequently amplified with the Syber Green One-Step PCR Master Kit in a 48-well plate (Applied Biosystems Inc.) as follows: 10 min at 95°C for enzyme activation, followed by 40 cycles of 15 s at 95°C, 20 s at 55°C, and 30 s at 72°C for the amplification step. Changes in the expression of each target gene were normalized relative to the mean critical threshold values of glyceraldehyde-3-phosphate dehydrogenase housekeeping gene by the 2−ΔΔCt method . We used 1 μl of both primers specific for each target gene. Primer sequence and annealing temperature specific for each gene are demonstrated in [Table 1].
|Table 1 Primer sequence and annealing temperature specific for each gene|
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All the data collected from the experiment were recorded and analyzed using IBM SPSS statistics software for Windows, version 20 (IBM Corp., Armonk, New York, USA). Paired t-test was used to compare differences between groups of neural function tests and one-way analysis of variance, with the post-hoc Scheffe’s test being used to compare differences among the groups of morphometric results and quantitative genes expression. In each test, the data were expressed as the mean value and SD, and differences were considered to be significant at P less than 0.01.
| Results|| |
Neural function results
All the data of the neural function evaluation tests (the righting reflex and the geotactic reflex) for all groups and at all time points are represented in [Table 2] and [Table 3], [Figure 1]a and [Figure 1]b. Group IIb at 28 days showed significant decrease (P<0.01) in reflexes time (recovery) compared with group IIb at 3 days. Group III (encephalopathy + stem cell group) showed significant decrease (P<0.01) in reflexes time at days 14 and 28 compared with group IIb.
|Table 2 Mean and SD of the righting reflex time in seconds at days 3, 7, 14, and 28 for all groups|
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|Table 3 Mean and SD of the geotactic reflex time in seconds at days 3, 7, 14, and 28 for all groups|
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|Figure 1 Line charts showing the mean of (a) righting reflex time and (b) geotactic reflex time (in seconds) at days 3, 7, 14, and 28 for all groups|
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Bone marrow-derived mesenchymal stem cells identification in culture and tracking
The BM-MSCs were identified after 2 weeks in culture with an inverted microscope as spindle-shaped cells between rounded cells ([Figure 2]a), and intravenously injected BM-MSCs labeled with green fluorescent protein were observed in cerebral cortex tissue using a fluorescent microscope ([Figure 2]b).
|Figure 2 Photomicrographs (×200) of bone marrow-derived mesenchymal stem cells showing (a) many spindle-shaped stem cells (curved arrow) on day 14 from primary culture (inverted microscope). (b) Bone marrow-derived mesenchymal stem cells labeled with green fluorescent protein (curved arrow) from stem cells group (fluorescent microscope)|
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Hematoxylin and eosin stain
Group I (groups Ia, Ib, and Ic) showed normal histological structure of the cerebral cortex, arranged in six layers ([Figure 3]a). The cerebral cortex contains pyramidal cells with large vesicular nuclei and prominent nucleoli, granule cells, neuroglia cells, and blood vessels ([Figure 3]b). Group IIa (encephalopathy group) showed many degenerated pyramidal cells and others appeared shrunken with a dark cytoplasm and small pyknotic nuclei. Excess vacuoles in neuropils and wide perineural space and perivascular space were noticed ([Figure 3]c). Group IIb (recovery) showed the same picture as group IIa except for the presence of few apparently normal pyramidal cells and many granule cells ([Figure 3]d). Group III (encephalopathy + stem cell group) showed many apparently normal pyramidal cells and excess granule cells. Few pyramidal cells appeared shrunken with slightly wide perineural space. Excess vacuoles in neuropils and narrow perivascular space were noticed ([Figure 3]e).
|Figure 3 Photomicrographs of a section in cerebral cortex stained with hematoxylin and eosin. (a) Group I (control group) showing the six layers: outer molecular layer (I), outer granular layer (II), outer pyramidal layer (III), inner granular layer (IV), inner pyramidal layer (V), and multiform layer (VI). (b) A higher magnification of the previous image showing pyramidal cells (P) with large nuclei, granule cells (G), neuroglia cells (arrow), and blood vessels (V). Narrow perineural space (*) and perivascular space (**) without obvious neuropil vacuolations were observed. (c) Group IIa (encephalopathy group) showing many degenerated (D) and other shrunken (K) pyramidal cells. Excess vacuoles (U) in neuropils, wide perineural space (*), and perivascular space (**) were noticed. (d) Group IIb (recovery group) showing many degenerated (D) and others shrunken (K) pyramidal cells, excess vacuoles (U) in neuropils and wide perineural space (*) and perivascular space (**). Few apparently normal pyramidal cells (P) and many granule cells (G) were observed. (e) Group III (encephalopathy + stem cell group) showing many apparently normal pyramidal cells (P) and excess granule cells (G). Few pyramidal cells appeared shrunken (K) with slightly wide perineural space (*). Excess vacuoles (U) in neuropils and narrow perivascular space (**) were noticed. (a) ×200; (b–e) ×400|
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Nestin immunostaining: nestin, a class VI intermediate filament protein, is expressed in adult neural progenitor cells. Nestin-immunoreactivity cells showed brown cytoplasmic staining. Nestin immunoexpression of all groups is represented in [Figure 4]a–[Figure 4]d.
|Figure 4 Photomicrographs of a section in cerebral cortex showing nestin immunoexpression (curved arrow), ×400. (a) Negative in control group. (b and c) Mild in groups IIa and IIb. (d) Marked in group III. (e) A histogram for the mean area percentage of nestin immunoexpression in groups I, IIa, IIb, and III|
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GFAP immunostaining: it is used to examine the distribution of astrocytes and their response to neural injury. GFAP-immunoreactivity astrocytes showed brown cytoplasmic staining together with their processes. GFAP immunoexpression of all groups is represented in [Figure 5]a–[Figure 5]d.
|Figure 5 Photomicrographs of a section in cerebral cortex showing glial fibrillary acidic protein immunoexpression (curved arrow), ×400. (a) Normal in control group. (b and c) Marked in groups IIa and IIb. (d) Near normal in group III. (e) A histogram for the mean area percentage of glial fibrillary acidic protein immunoexpression in groups I, IIa, IIb, and III|
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NFs immunostaining: NFs were expressed in somal and axonal membranes as brown staining particles. NF immunoexpression of all groups is represented in [Figure 6]a–[Figure 6]d.
|Figure 6 Photomicrographs of a section in cerebral cortex showing neurofilament immunoexpression (curved arrow), ×400. (a) Normal (marked) in control group. (b and c) Mild in groups IIa and IIb. (d) Near normal in group III. (e) A histogram for the mean area percentage of neurofilament immunoexpression in groups I, IIa, IIb, and III|
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The mean area percentage and SD of nestin, GFAP, and NF immunoexpression for all groups are represented in [Table 4],[Table 5],[Table 6], Figs 4h, 5h and 6h. There was a significant increase in mean area percentage of nestin and NF immunoexpression and a significant decrease in GFAP immunoexpression of group III (encephalopathy + stem cell group) compared with group IIa. Group IIb (recovery) showed insignificant changes in mean area percentage of nestin, GFAP, and NF immunoexpression compared with group IIa.
|Table 4 Mean area percentage and SD of nestin immunoexpression for all groups, with comparison between all groups by post-hoc Scheffe’s test|
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|Table 5 Mean area percentage and SD of glial fibrillary acidic protein immunoexpression for all groups, with comparison between all groups by post-hoc Scheffe’s test|
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|Table 6 Mean area percentage and SD of neurofilaments immunoexpression for all groups, with comparison between all groups by post-hoc Scheffe’s test|
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Quantitative genes expression
All the data of VEGF-R2 and eNOS gene expression for all groups are represented in [Table 7] and [Figure 7]. Compared with group IIa, group III showed significant increase in mean gene expression of both genes whereas there were insignificant changes in group IIb.
|Table 7 Mean and SD of vascular endothelial growth factor-receptor 2 and endothelial nitric oxide synthase genes expression for all groups, with comparison between groups by post-hoc Scheffe’s test|
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|Figure 7 A histogram for the mean of vascular endothelial growth factor-receptor 2 and endothelial nitric oxide synthase genes expression for all groups|
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| Discussion|| |
HIE caused by perinatal or neonatal hypoxia is still a major global cause of neonatal mortality and morbidity even in developed countries .
Rice method (unilateral ligation of carotid artery in a 7-day-old rat followed by 2.5–3 h of low oxygen concentration treatment) is currently the most commonly used method for the preparation of HIE animal model . Cerebral blood supply of rats is similar to humans; internal carotid and vertebral arteries form Willis ring on the bottom of the brain, which can communicate and compensate on both side, and unilateral ligation of carotid artery cannot cause marked brain injury . So, induction of HI brain injuries of rats in the present study was performed through bilateral cephalic arteries ligation after ether anesthesia, and rats were placed in the container aerated with 8% O2 and 92% N2 for 1.5 h at 37°C. Induction of HIE in the present study was performed in a 7-day-old rat because this is the age of peak brain growth, which occurs at term in humans .
Rats in group IIa (encephalopathy group) in the present study were killed 3 days after induction of HI brain injuries to study the histopathological changes in cerebral cortex as brain structure disorder appeared in the cerebral cortex 3 h after HIE, and the histopathological changes continued to worsen after 3 days . Also, some researchers  reported that brain injury (cell degeneration and necrosis) became obvious 24 h after HIE and aggravated after 48 h, and large areas of cellular liquefaction necrosis in the brain tissue of rats were found at 96 h that maybe extended to 7 days.
Group IIa in the present study showed prolonged reflexes time in neural function evaluation, marked injury of cerebral cortex, mild nestin and NF immunoexpression, marked GFAP immunoexpression, and VEGF-R2 and eNOS genes expression. Group IIb (recovery) in the present study showed nearly the same picture as group IIa and insignificant changes in mean area percentage of nestin, GFAP, and NF immunoexpression and in mean gene expression compared with group IIa. Only, group IIb at 28 days showed significant decrease (P<0.01) in reflexes time (recovery) compared with group IIb at 3 days. In agreement with these findings, many studies ,, showed that neonatal HIE resulted in neuronal and axonal injury and also impaired neurobehavioral performance in rats. A previous study  showed that rats showed longer mean latency time (righting reflex) as compared with the control group from 1 to 11 days after induction of HIE, and recovery from this impairment started on day 12. Another study  showed that astrocytes of cerebral cortex showed strong GFAP-positive fibers 1 week after the bilateral common carotid arteries occlusion in rats. The immunoreactivity had returned to control levels in gray matter areas after 2 months of bilateral common carotid arteries occlusion, whereas the fiber tracts remained elevated after 4 months. Many researchers , showed in their studies that at day 1, cerebral cortex showed nestin immunoexpression (a marker of undifferentiated neural progenitors) that continued to increase, peaked at day 5, and began to decrease after day 7. However, a previous study  demonstrated that nestin immunoexpression may be persisted up to 6 months in some areas of cerebral cortex. Van den Tweel et al.  reported that HI in the neonatal rat brain induced a transient increase in eNOS expression as a neuroprotective mechanism. Greenberg and Jin  reported that cerebral ischemia increased VEGFR-2 expression in endothelial cells and astrocytes, which were detectable at 1–3 days. VEGFs including VEGFR-2 have been implicated in hypoxia-driven sprouting of new capillaries from existing vessels. So, the improvement in neural function evaluation tests at day 28 in group IIb of the present study may be because of relieve of the brain edema as ischemia and hypoxia increase cerebral edema, which aggravate brain damage .
Though a primary cause of HI injury is decreasing the oxygen supply to neonatal brain tissues, the mechanisms of subsequent events that occurred in HIE are less understood . However, many studies ,,,,, explained the neonatal brain injury is induced by HIE. Brain damage following a neonatal HIE is a multiple subsequent process and involved two phases. First phase is the early energy failure phase, where the oxidative energy metabolism of cells decreases leading to necrotic death with subsequent moreover injury in the second phase, which is the late energy failure phase. The secondary phase occurs from 6 to 48 h after the initial injury and appears to be related to a cascade of biochemical events, which include production of oxygen free radicals, inflammation, cell membrane disturbances, and apoptosis, among others. Oxidative stress is particularly injurious to the neonatal brain owing to low antioxidants concentrations and increased oxygen consumption at the period of transformation from prenatal to postnatal life. Dixon et al.  postulated that there is a third phase where harmful agents cause more damage and worsen the results. This third phase includes mechanisms of inflammation and epigenetic changes that lead to an impairment or alteration of neuronal formation and growth.
In spite of the large morbidity and mortality associated with HIE, many therapeutic modalities including erythropoietin administration and therapeutic hypothermia have several complications and limitations ,.
Group II (encephalopathy + stem cell group) in the present study showed improvement in all parameters (neural function tests, histopathological studies, and genes expression) compared with encephalopathy group. In agreement with these findings, one study  reported that BM-MSCs have been used in the treatment of neonatal HIE and show reduction in sensorimotor and memory impairments. Some researchers  reported that MSCs showed potent regenerative effects on HI brain damage in the neonatal mouse as MSCs decrease the glial scar formation. Other researchers  reported that MSCs transplantation improved the functional neural tests, reduced the histologic abnormalities, and significantly decreased the GFAP expression in ischemic brain injury of newborn rats.
Stem cell-mediated functional recovery in HI brain injury through two major actions: cell replacement and paracrine effect . MSCs are able to migrate to the site of injury, differentiate into specific lineages, aid brain tissue repair through possible replacement of damaged neurons and oligodendrocytes, and modulate host inflammatory response . The paracrine action of MSCs facilitates the repair process made by the parenchymal cells of the recipient, and such processes may include blood vessel regeneration, replacement of damaged nerve cells, and improved survival of intrinsic neuronal cells. The repair process is facilitated by a significant dampening of inflammatory pathways . IH increases stem cell survival and the secretion of VEGF .
| Conclusion|| |
BM-MSCs attenuated HIE in rats, but compared with the normal control rats, this effect was still away. However, further researches are essential to evaluate the efficacy of combination therapy between stem cells and other therapeutic agents such as antioxidants in the treatment of HIE.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7]