Exosomes Treatment Mitigates Ischemic Brain Damage but Does Not Improve Post-Stroke Neurological Outcome.

BACKGROUND/AIMS: Recent studies demonstrated that the treatment with mesenchymal stem cells (MSCs) obtained from the human umbilical cord blood improved survival, reduced brain damage, prevented apoptosis, suppressed inflammatory responses, downregulated the DNA damage-inducing genes, upregulated the DNA repair genes, and facilitated neurological recovery in stroke-induced animals. Emerging stroke literature supports the concept that the exosomes released from MSCs are the primary biological principles underlying the post-stroke neuroprotection offered by MSCs treatment. METHODS: Because the treatment with exosomes has a great potential to overcome the limitations associated with cell-based therapies, we tested the efficacy of exosomes secreted from HUCB-MSCs under standard culture conditions on post-stroke brain damage and neurological outcome in a rat model of ischemic stroke by performing TTC staining as well as the modified neurological severity scores, modified adhesive removal, beam-walking, and accelerating Rotarod performance tests before ischemia and at regular intervals until seven days reperfusion. RESULTS: Exosomes treatment attenuated the infarct size. Treatment with exosomes did not affect the post-stroke survival rate and body weight changes, but exacerbated the somatosensory and motor dysfunction and adversely affected the natural recovery that occurs without any treatment. CONCLUSION: Treatment with exosomes secreted from HUCB-MSCs under standard culture conditions attenuates the ischemic brain damage but does not improve the post-stroke neurological outcome.

Mesenchymal Stem Cell Treatment Prevents Post-Stroke Dysregulation of Matrix Metalloproteinases and Tissue Inhibitors of Metalloproteinases.

BACKGROUND/AIMS: Stem cell treatment is one of the potential treatment options for ischemic stroke. We recently demonstrated a protective effect of human umbilical cord blood-derived mesenchymal stem cells (HUCB-MSCs) in a rat model of ischemic stroke. The treatment attenuated apoptosis and prevented DNA damage. A collection of published studies, including several from our laboratory, indicated the induction and detrimental role for several matrix metalloproteinases (MMPs) in post-stroke brain injury. We hypothesized that the HUCB-MSCs treatment after focal cerebral ischemia prevents the dysregulation of MMPs and induces the expression of endogenous tissue inhibitors of metalloproteinases (TIMPs) to neutralize the elevated activity of MMPs. METHODS: To test our hypothesis, we administered HUCB-MSCs (0.25 million cells/animal and 1 million cells/animal) intravenously via tail vein to male Sprague-Dawley rats that were subjected to a transient (two-hour) right middle cerebral artery occlusion (MCAO) and one-day reperfusion. Ischemic brain tissues obtained from various groups of rats seven days after reperfusion were subjected to real-time PCR, immunoblot, and immunofluorescence analysis. RESULTS: HUCB-MSCs treatment prevented the induction of MMPs, which were upregulated in ischemia-induced rats that received no treatment. HUCB-MSCs treatment also prevented the induction of TIMPs expression. The extent of prevention of MMPs and TIMPs induction by HUCB-MSCs treatment is similar at both the doses tested. CONCLUSION: Prevention of stroke-induced MMPs upregulation after HUCB-MSCs treatment is not mediated through TIMPs upregulation.

Matrix Metalloproteinase-12 Induces Blood-Brain Barrier Damage After Focal Cerebral Ischemia.

BACKGROUND AND PURPOSE: Matrix metalloproteinases (MMPs) have a central role in compromising the integrity of the blood-brain barrier (BBB). The role of MMP-12 in brain damage after ischemic stroke remains unknown. The main objective of the current study is to investigate the effect of MMP-12 suppression at an early time point before reperfusion on the BBB damage in rats. METHODS: Sprague-Dawley rats were subjected to middle cerebral artery occlusion and reperfusion. MMP-12 shRNA-expressing plasmids formulated as nanoparticles were administered at a dose of 1 mg/kg body weight. The involvement of MMP-12 on BBB damage was assessed by performing various techniques, including Evans blue dye extravasation, 2,3,5-triphenyltetrazolium chloride staining, immunoblot, gelatin zymography, and immunofluorescence analysis. RESULTS: MMP-12 is upregulated ≈31-, 47-, and 66-fold in rats subjected 1-, 2-, or 4-hour ischemia, respectively, followed by 1-day reperfusion. MMP-12 suppression protected the BBB integrity by inhibiting the degradation of tight-junction proteins. Either intravenous or intra-arterial delivery of MMP-12 shRNA-expressing plasmid significantly reduced the percent Evans blue dye extravasation and infarct size. Furthermore, MMP-12 suppression reduced the endogenous levels of other proteases, such as tissue-type plasminogen activator and MMP-9, which are also known to be the key players involved in BBB damage. CONCLUSIONS: These results demonstrate the adverse role of MMP-12 in acute brain damage that occurs after ischemic stroke and, thereby, suggesting that MMP-12 suppression could be a promising therapeutic target for cerebral ischemia.

Stem cell treatment after cerebral ischemia regulates the gene expression of apoptotic molecules.

Evidence suggests that apoptosis contributes significantly to cell death after cerebral ischemia. Our recent studies that utilized human umbilical cord blood-derived mesenchymal stem cells (hUCBSCs) demonstrated the potential of hUCBSCs to inhibit neuronal apoptosis in a rat model of CNS injury. Therefore, we hypothesize that intravenous administration of hUCBSCs after focal cerebral ischemia would reduce brain damage by inhibiting apoptosis and downregulating the upregulated apoptotic pathway molecules. Male Sprague-Dawley rats were obtained and randomly assigned to various groups. After the animals reached a desired weight, they were subjected to a 2 h middle cerebral artery occlusion (MCAO) procedure followed by 7 days of reperfusion. The hUCBSCs were obtained, cultured, and intravenously injected (0.25 × 10(6) cells or 1 × 10(6) cells) via the tail vein to separate groups of animals 24 h post-MCAO procedure. We performed various techniques including PCR microarray, hematoxylin and eosin, and TUNEL staining in addition to immunoblot and immunofluorescence analysis in order to investigate the effect of our treatment on regulation of apoptosis after focal cerebral ischemia. Most of the apoptotic pathway molecules which were upregulated after focal cerebral ischemia were downregulated after hUCBSCs treatment. Further, the staining techniques revealed a prominent reduction in brain damage and the extent of apoptosis at even the lowest dose of hUCBSCs tested in the present study. In conclusion, our treatment with hUCBSCs after cerebral ischemia in the rodent reduces brain damage by inhibiting apoptosis and downregulating the apoptotic pathway molecules.

The paradox of tPA in ischemic stroke: tPA knockdown following recanalization improves functional and histological outcomes.

Previous studies have demonstrated that endogenous tissue-type plasminogen activator (tPA) is upregulated in the brain after an acute ischemic stroke (AIS). While mixed results were observed in genetic models, the pharmacological inhibition of endogenous tPA showed beneficial effects. Treatment with exogenous recombinant tPA exacerbated brain damage in rodent models of stroke. Despite the detrimental effects of tPA in ischemic stroke, recombinant tPA is administered to AIS patients to recanalize the occluded blood vessels because the benefits of its administration outweigh the risks associated with tPA upregulation and increased activity. We hypothesized that tPA knockdown following recanalization would ameliorate sensorimotor deficits and reduce brain injury. Young male and female rats (2-3 months old) were subjected to transient focal cerebral ischemia by occlusion of the right middle cerebral artery. Shortly after reperfusion, rats from appropriate cohorts were administered a nanoparticle formulation containing tPA shRNA or control shRNA plasmids (1 mg/kg) intravenously via the tail vein. Infarct volume during acute and chronic phases, expression of matrix metalloproteinases (MMPs) 1, 3, and 9, enlargement of cerebral ventricle volume, and white matter damage were all reduced by shRNA-mediated gene silencing of tPA following reperfusion. Additionally, recovery of somatosensory and motor functions was improved. In conclusion, our results provide evidence that reducing endogenous tPA following recanalization improves functional outcomes and reduces post-stroke brain damage.

Involvement of nitric oxide synthase in matrix metalloproteinase-9- and/or urokinase plasminogen activator receptor-mediated glioma cell migration.

BACKGROUND: Src tyrosine kinase activates inducible nitric oxide synthase (iNOS) and, in turn, nitric oxide production as a means to transduce cell migration. Src tyrosine kinase plays a key proximal role to control α9ß1 signaling. Our recent studies have clearly demonstrated the role of α9ß1 integrin in matrix metalloproteinase-9 (MMP-9) and/or urokinase plasminogen activator receptor (uPAR)-mediated glioma cell migration. In the present study, we evaluated the involvement of α9ß1 integrin-iNOS pathway in MMP-9- and/or uPAR-mediated glioma cell migration. METHODS: MMP-9 and uPAR shRNAs and overexpressing plasmids were used to downregulate and upregulate these molecules, respectively in U251 glioma cells and 5310 glioma xenograft cells. The effect of treatments on migration and invasion potential of these glioma cells were assessed by spheroid migration, wound healing, and Matrigel invasion assays. In order to attain the other objectives we also performed immunocytochemical, immunohistochemical, RT-PCR, Western blot and fluorescence-activated cell sorting (FACS) analysis. RESULTS: Immunohistochemical analysis revealed the prominent association of iNOS with glioblastoma multiforme (GBM). Immunofluorescence analysis showed prominent expression of iNOS in glioma cells. MMP-9 and/or uPAR knockdown by respective shRNAs reduced iNOS expression in these glioma cells. RT-PCR analysis revealed elevated iNOS mRNA expression in either MMP-9 or uPAR overexpressed glioma cells. The migration potential of MMP-9- and/or uPAR-overexpressed U251 glioma cells was significantly inhibited after treatment with L-NAME, an inhibitor of iNOS. Similarly, a significant inhibition of the invasion potential of the control or MMP-9/uPAR-overexpressed glioma cells was noticed after L-NAME treatment. A prominent reduction of iNOS expression was observed in the tumor regions of nude mice brains, which were injected with 5310 glioma cells, after MMP-9 and/or uPAR knockdown. Protein expressions of cSrc, phosphoSrc and p130Cas were reduced with simultaneous knockdown of both MMP-9 and uPAR. CONCLUSIONS: Taken together, our results from the present and earlier studies clearly demonstrate that α9ß1 integrin-mediated cell migration utilizes the iNOS pathway, and inhibition of the migratory potential of glioma cells by simultaneous knockdown of MMP-9 and uPAR could be attributed to the reduced α9ß1 integrin and iNOS levels.

Implications of MMP-12 in the pathophysiology of ischaemic stroke.

This article focuses on the emerging role of matrix metalloproteinase-12 (MMP-12) in ischaemic stroke (IS). MMP-12 expression in the brain increases dramatically in animal models of IS, and its suppression reduces brain damage and promotes neurological, sensorimotor and cognitive functional outcomes. Thus, MMP-12 could represent a potential target for the management of IS. This article provides an overview of MMP-12 upregulation in the brain following IS, its deleterious role in the post-stroke pathogenesis (blood-brain barrier disruption, inflammation, apoptosis and demyelination), possible molecular interactions and mechanistic insights, its involvement in post-ischaemic functional deficits and recovery as well as the limitations, perspectives, challenges and future directions for further research. Prior to testing any MMP-12-targeted therapy in patients with acute IS, additional research is needed to establish the effectiveness of MMP-12 suppression against IS in older animals and in animals with comorbidities. This article also examines the clinical implications of suppressing MMP-12 alone or in combination with MMP-9 for extending the currently limited tissue plasminogen activator therapy time window. Targeting of MMP-12 is expected to have a profound influence on the therapeutic management of IS in the future.

The Impact of Social Isolation and Environmental Deprivation on Blood Pressure and Depression-Like Behavior in Young Male and Female Mice.

Background: Social isolation (SI) and loneliness are major adult and adolescent health concerns, particularly in the coronavirus disease 2019 (COVID-19) era. Recent prospective cohort studies indicate that older women who experienced both SI and loneliness had a significantly higher risk of cardiovascular disease (CVD). Hypertension, a well-established risk factor for CVD, is more prevalent in elderly women than men. Furthermore, a lack of social relationships is strongly associated with an increased risk of hypertension in middle-aged and elderly women compared to men. Although this has not been extensively studied, adolescents and young adults who experience loneliness or SI may also be at risk for CVD and depression. The purpose of this study was to examine the effect of SI on blood pressure and depression-like behavior in young male and female mice. Methods: Weaned C57BL/6 mice were randomly assigned (n = 6/group/sex) to either group housing (GH) or SI. Animals in the SI group were housed in individual cages for 8 weeks with no view of other animals. The cages were kept in ventilated racks to prevent pheromone exposure and socially isolated animals had no cage enrichment. Results: SI increased systolic, diastolic, and mean arterial blood pressure in females and elevated heart rate in both sexes. Body weight gain was dramatically increased in socially isolated females but tended to decrease in socially isolated males. In the forced swim test, which detects depression-like behavior, there was no difference between groups in total immobility time. The latency to immobility, however, was significantly decreased in socially isolated females. Serum concentrations of corticosterone and metanephrine did not differ between socially isolated and group-housed females, but corticosterone levels were significantly reduced in socially isolated males. Conclusions: Our results indicate that 8 weeks of SI leads to significant changes in blood pressure and heart rate and mild changes in depression-like behavior in young mice, with females affected more than males.

MMP-12 knockdown prevents secondary brain damage after ischemic stroke in mice.

We previously reported that increased expression of matrix metalloproteinase-12 (MMP-12) mediates blood-brain barrier disruption via tight junction protein degradation after focal cerebral ischemia in rats. Currently, we evaluated whether MMP-12 knockdown protects the post-stroke mouse brain and promotes better functional recovery. Adult male mice were injected with negative siRNA or MMP-12 siRNA (intravenous) at 5 min of reperfusion following 1 h transient middle cerebral artery occlusion. MMP-12 knockdown significantly reduced the post-ischemic infarct volume and improved motor and cognitive functional recovery. Mechanistically, MMP-12 knockdown ameliorated degradation of tight junction proteins zonula occludens-1, claudin-5, and occludin after focal ischemia. MMP-12 knockdown also decreased the expression of inflammatory mediators, including monocyte chemoattractant protein-1, tumor necrosis factor-α, and interleukin-6, and the expression of apoptosis marker cleaved caspase-3 after ischemia. Overall, the present study indicates that MMP-12 promotes secondary brain damage after stroke and hence is a promising stroke therapeutic target.

Therapeutic efficacy of matrix metalloproteinase-12 suppression on neurological recovery after ischemic stroke: Optimal treatment timing and duration.

We recently showed that the post-ischemic induction of matrix metalloproteinase-12 (MMP-12) in the brain degrades tight junction proteins, increases MMP-9 and TNFα expression, and contributes to the blood-brain barrier (BBB) disruption, apoptosis, demyelination, and infarct volume development. The objectives of this study were to (1) determine the effect of MMP-12 suppression by shRNA-mediated gene silencing on neurological/functional recovery, (2) establish the optimal timing of MMP-12shRNA treatment that provides maximum therapeutic benefit, (3) compare the effectiveness of acute versus chronic MMP-12 suppression, and (4) evaluate potential sex-related differences in treatment outcomes. Young male and female Sprague-Dawley rats were subjected to transient middle cerebral artery occlusion and reperfusion. Cohorts of rats were administered either MMP-12shRNA or scrambled shRNA sequence (control) expressing plasmids (1 mg/kg; i.v.) formulated as nanoparticles. At designated time points after reperfusion, rats from various groups were subjected to a battery of neurological tests to assess their reflex, balance, sensory, and motor functions. Suppression of MMP-12 promoted the neurological recovery of stroke-induced male and female rats, although the effect was less apparent in females. Immediate treatment after reperfusion resulted in a better recovery of sensory and motor function than delayed treatments. Chronic MMP-12 suppression neither enhanced nor diminished the therapeutic effects of acute MMP-12 suppression, indicating that a single dose of plasmid may be sufficient. We conclude that suppressing MMP-12 after an ischemic stroke is a promising therapeutic strategy for promoting the recovery of neurological function.

The interplay between MMP-12 and t-PA in the brain after ischemic stroke.

Tissue-type plasminogen activator (t-PA) expression is known to increase following transient focal cerebral ischemia and reperfusion. Previously, we reported downregulation of t-PA upon suppression of matrix metalloproteinase-12 (MMP-12), following transient focal cerebral ischemia and reperfusion. We now present data on the temporal expression of t-PA in the brain after transient ischemia, as well as the interaction between MMP-12 and t-PA, two proteases associated with the breakdown of the blood-brain barrier (BBB) and ischemic brain damage. We hypothesized that there might be reciprocal interactions between MMP-12 and t-PA in the brain after ischemic stroke. This hypothesis was tested using shRNA-mediated gene silencing and computational modeling. Suppression of t-PA following transient ischemia and reperfusion in rats attenuated MMP-12 expression in the brain. The overall effect of t-PA shRNA administration was to attenuate the degradation of BBB tight junction protein claudin-5, diminish BBB disruption, and reduce neuroinflammation by decreasing the expression of the microglia/macrophage pro-inflammatory M1 phenotype (CD68, iNOS, IL-1ß, and TNFα). Reduced BBB disruption and subsequent lack of infiltration of macrophages (the main source of MMP-12 in the ischemic brain) could account for the decrease in MMP-12 expression after t-PA suppression. Computational modeling of in silico protein-protein interactions indicated that MMP-12 and t-PA may interact physically. Overall, our findings demonstrate that MMP-12 and t-PA interact directly or indirectly at multiple levels in the brain following an ischemic stroke. The present findings could be useful in the development of new pharmacotherapies for the treatment of stroke.

p53- and Bax-mediated apoptosis in injured rat spinal cord.

Spinal cord injury (SCI) induces a series of endogenous biochemical changes that lead to secondary degeneration, including apoptosis. p53-mediated mitochondrial apoptosis is likely to be an important mechanism of cell death in spinal cord injury. However, the signaling cascades that are activated before DNA fragmentation have not yet been determined. DNA damage-induced, p53-activated neuronal cell death has already been identified in several neurodegenerative diseases. To determine DNA damage-induced, p53-mediated apoptosis in spinal cord injury, we performed RT-PCR microarray and analyzed 84 DNA damaging and apoptotic genes. Genes involved in DNA damage and apoptosis were upregulated whereas anti-apoptotic genes were downregulated in injured spinal cords. Western blot analysis showed the upregulation of DNA damage-inducing protein such as ATM, cell cycle checkpoint kinases, 8-hydroxy-2'-deoxyguanosine (8-OHdG), BRCA2 and H2AX in injured spinal cord tissues. Detection of phospho-H2AX in the nucleus and release of 8-OHdG in cytosol were demonstrated by immunohistochemistry. Expression of p53 was observed in the neurons, oligodendrocytes and astrocytes after spinal cord injury. Upregulation of phospho-p53, Bax and downregulation of Bcl2 were detected after spinal cord injury. Sub-cellular distribution of Bax and cytochrome c indicated mitochondrial-mediated apoptosis taking place after spinal cord injury. In addition, we carried out immunohistochemical analysis to confirm Bax translocation into the mitochondria and activated p53 at Ser³9². Expression of APAF1, caspase 9 and caspase 3 activities confirmed the intrinsic apoptotic pathway after SCI. Activated p53 and Bax mitochondrial translocation were detected in injured spinal neurons. Taken together, the in vitro data strengthened the in vivo observations of DNA damage-induced p53-mediated mitochondrial apoptosis in the injured spinal cord.

[Retracted] Metabolic remodeling precedes mitochondrial outer membrane permeabilization in human glioma xenograft cells.

Following the publication of the above paper, we were contacted by the University of Illinois at Chicago, to request the retraction of the above article. Following a formal institutional investigation, the investigation panel concluded that the images in question had falsifying elements. Regarding the above study, the specific allegations that were investigated were that of falsifying elements of Fig. 6A, row 2, columns 2 and 3. Following a review of this paper conducted independently by the Editor of International Journal of Oncology, the Editor concurred with the conclusions of the investigation panel, and therefore the above paper has been retracted from the publication. We also tried to contact the authors, but did not receive a reply. The Editor apologizes to the readership for the inconvenience caused.[the original article was published in International Journal of Oncology 40: 509­518, 2012; DOI: 10.3892/ijo.2011.1255].

Stem cell treatment improves post stroke neurological outcomes: a comparative study in male and female rats.

BACKGROUND AND PURPOSE: The therapeutic potential of different stem cells for ischaemic stroke treatment is intriguing and somewhat controversial. Recent results from our laboratory have demonstrated the potential benefits of human umbilical cord blood-derived mesenchymal stem cells (MSC) in a rodent stroke model. We hypothesised that MSC treatment would effectively promote the recovery of sensory and motor function in both males and females, despite any apparent sex differences in post stroke brain injury. METHODS: Transient focal cerebral ischaemia was induced in adult Sprague-Dawley rats by occlusion of the middle cerebral artery. Following the procedure, male and female rats of the untreated group were euthanised 1 day after reperfusion and their brains were used to estimate the resulting infarct volume and tissue swelling. Additional groups of stroke-induced male and female rats were treated with MSC or vehicle and were subsequently subjected to a battery of standard neurological/neurobehavioral tests (Modified Neurological Severity Score assessment, adhesive tape removal, beam walk and rotarod). The tests were administered at regular intervals (at days 1, 3, 5, 7 and 14) after reperfusion to determine the time course of neurological and functional recovery after stroke. RESULTS: The infarct volume and extent of swelling of the ischaemic brain were similar in males and females. Despite similar pathological stroke lesions, the clinical manifestations of stroke were more pronounced in males than females, as indicated by the neurological scores and other tests. MSC treatment significantly improved the recovery of sensory and motor function in both sexes, and it demonstrated efficacy in both moderate stroke (females) and severe stroke (males). CONCLUSIONS: Despite sex differences in the severity of post stroke outcomes, MSC treatment promoted the recovery of sensory and motor function in male and female rats, suggesting that it may be a promising treatment for stroke.

Attenuation of the Induction of TLRs 2 and 4 Mitigates Inflammation and Promotes Neurological Recovery After Focal Cerebral Ischemia.

The intense inflammatory response triggered in the brain after focal cerebral ischemia is detrimental. Recently, we showed that the suppression of toll-like receptors (TLRs) 2 and 4 attenuates infarct size and reduces the expression of pro-inflammatory cytokines in the ischemic brain. In this study, we further examined the effect of unsuppressed induction of TLRs 2 and 4 on the expression of its downstream signaling molecules and pro-inflammatory cytokines 1 week after reperfusion. The primary purpose of this study was to investigate the effect of simultaneous knockdown of TLRs 2 and 4 on M1/M2 microglial polarization dynamics and post-stroke neurological deficits and the recovery. Transient focal cerebral ischemia was induced in young adult male Sprague-Dawley rats by the middle cerebral artery occlusion (MCAO) procedure using a monofilament suture. Appropriate cohorts of rats were treated with a nanoparticle formulation of TLR2shRNA and TLR4shRNA (T2sh+T4sh) expressing plasmids (1 mg/kg each of T2sh and T4sh) or scrambled sequence inserted vector (vehicle control) expressing plasmids (2 mg/kg) intravenously via tail vein immediately after reperfusion. Animals from various cohorts were euthanized during reperfusion, and the ischemic brain tissue was isolated and utilized for PCR followed by agarose gel electrophoresis, real-time PCR, immunoblot, and immunofluorescence analysis. Appropriate groups were subjected to a battery of standard neurological tests at regular intervals until 14 days after reperfusion. The increased expression of both TLRs 2 and 4 and their downstream signaling molecules including the pro-inflammatory cytokines was observed even at 1-week after reperfusion. T2sh+T4sh treatment immediately after reperfusion attenuated the post-ischemic inflammation, preserved the motor function, and promoted recovery of the sensory and motor functions. We conclude that the post-ischemic induction of TLRs 2 and 4 persists for at least 7 days after reperfusion, contributes to the severity of acute inflammation, and impedes neurological recovery. Unlike previous studies in TLRs 2 or 4 knockout models, results of this study in a pharmacologically relevant preclinical rodent stroke model have translational significance.

Human umbilical cord blood stem cells upregulate matrix metalloproteinase-2 in rats after spinal cord injury.

Matrix metalloproteinases (MMPs) are a large family of proteolytic enzymes involved in inflammation, wound healing and other pathological processes after neurological disorders. MMP-2 promotes functional recovery after spinal cord injury (SCI) by regulating the formation of a glial scar. In the present study, we aimed to investigate the expression and/or activity of several MMPs, after SCI and human umbilical cord blood mesenchymal stem cell (hUCB) treatment in rats with a special emphasis on MMP-2. Treatment with hUCB after SCI altered the expression of several MMPs in rats. MMP-2 is upregulated after hUCB treatment in spinal cord injured rats and in spinal neurons injured either with staurosporine or hydrogen peroxide. Further, hUCB induced upregulation of MMP-2 reduced formation of the glial scar at the site of injury along with reduced immunoreactivity to chondroitin sulfate proteoglycans. Blockade of MMP-2 activity in hUCB cocultured injured spinal neurons reduced the protection offered by hUCB which indicated the involvement of MMP-2 in the neuroprotection offered by hUCB. Based on these results, we conclude that hUCB treatment after SCI upregulates MMP-2 levels and reduces the formation of the glial scar thereby creating an environment suitable for endogenous repair mechanisms.

Stem cells downregulate the elevated levels of tissue plasminogen activator in rats after spinal cord injury.

We investigated the involvement of tPA after SCI in rats and effect of treatment with human umbilical cord blood derived stem cells. tPA expression and activity were determined in vivo after SCI in rats and in vitro in rat embryonic spinal neurons in response to injury with staurosporine, hydrogen peroxide and glutamate. The activity and/or expression of tPA increased after SCI and reached peak levels on day 21 post-SCI. Notably, the tPA mRNA activity was upregulated by 310-fold compared to controls on day 21 post-SCI. As expected, MBP expression is minimal at the time of peak tPA activity and vice versa. Implantation of hUCB after SCI resulted in the downregulation of elevated tPA activity/expression in vivo in rats as well as in vitro in spinal neurons. Our results demonstrated the involvement of tPA in the secondary pathogenesis after SCI as well as the therapeutic potential of hUCB.

Exosomes Secreted by the Cocultures of Normal and Oxygen-Glucose-Deprived Stem Cells Improve Post-stroke Outcome.

Emerging stroke literature suggests that treatment of experimentally induced stroke with stem cells offered post-stroke neuroprotection via exosomes produced by these cells. Treatment with exosomes has great potential to overcome the limitations associated with cell-based therapies. However, in our preliminary studies, we noticed that the exosomes released from human umbilical cord blood-derived mesenchymal stem cells (MSCs) under standard culture conditions did not improve the post-stroke neurological outcome. Because of this apparent discrepancy, we hypothesized that exosome characteristics vary with the conditions of their production. Specifically, we suggest that the exosomes produced from the cocultures of regular and oxygen-glucose-deprived (OGD) MSCs in vitro would represent the exosomes produced from MSCs that are exposed to ischemic brain cells in vivo, and offer similar therapeutic benefits that the cell treatment would provide. We tested the efficacy of therapy with exosomes secreted from human umbilical cord blood (HUCB)-derived MSCs under in vitro hypoxic conditions on post-stroke brain damage and neurological outcome in a rat model of transient focal cerebral ischemia. We performed the TTC staining procedure as well as the neurological tests including the modified neurological severity scores (mNSS), the modified adhesive removal (sticky-tape), and the beam walking tests before ischemia and at regular intervals until 7 days reperfusion. Treatment with exosomes obtained from the cocultures of normal and OGD-induced MSCs reduced the infarct size and ipsilateral hemisphere swelling, preserved the neurological function, and facilitated the recovery of stroke-induced rats. Based on the results, we conclude that the treatment with exosomes secreted from MSCs at appropriate experimental conditions attenuates the post-stroke brain damage and improves the neurological outcome.

Neuroprotection by cord blood stem cells against glutamate-induced apoptosis is mediated by Akt pathway.

The neurotransmitter glutamate mediates excitatory synaptic transmission in the brain and spinal cord. In pathological conditions massive glutamate release reaches near millimolar concentrations in the extracellular space and contributes to neuron degeneration and death. In the present study, we demonstrate a neuroprotective role for human umbilical cord blood stem cells (hUCB) against glutamate-induced apoptosis in cultured rat cortical neurons. Microarray analysis shows the upregulation of stress pathway genes after glutamate toxicity of neurons, while in cocultures with hUCB, survival pathway genes were upregulated. Real time-PCR analysis shows the expression of genes for NMDA receptors after glutamate toxicity in neurons. The neuroprotection of hUCB against glutamate toxicity is similar to the application of the glutamate receptor antagonist MK-801. Cocultures of hUCB protected neurons against glutamate-induced apoptosis as revealed by APO-BrdU TUNEL and FACS analyses. Immunoblot analysis shows that apoptosis is mediated by the cleavage of caspase-3 and caspase-7 in glutamate treated neurons. Cocultures with hUCB indicate the upregulation of Akt signaling pathway to protect neurons. Blocking of the Akt pathway by a dominant-negative Akt and using Akt-inhibitor IV, we confirm that the mechanism underlying hUCB neuroprotection involves activation of Akt signaling pathway. These results suggest the neuroprotective potential of hUCB against glutamate-induced apoptosis of cultured cortical neurons.