Yanlu Zhang

Effect of exosomes derived from multipluripotent mesenchymal stromal cells on functional recovery and neurovascular plasticity in rats after traumatic brain injury.

OBJECT Transplanted multipotent mesenchymal stromal cells (MSCs) improve functional recovery in rats after traumatic brain injury (TBI). In this study the authors tested a novel hypothesis that systemic administration of cell-free exosomes generated from MSCs promotes functional recovery and neurovascular remodeling in rats after TBI. METHODS Two groups of 8 Wistar rats were subjected to TBI, followed 24 hours later by tail vein injection of 100 μg protein of exosomes derived from MSCs or an equal volume of vehicle (phosphate-buffered saline). A third group of 8 rats was used as sham-injured, sham-treated controls. To evaluate cognitive and sensorimotor functional recovery, the modified Morris water maze, modified Neurological Severity Score, and foot-fault tests were performed. Animals were killed at 35 days after TBI. Histopathological and immunohistochemical analyses were performed for measurements of lesion volume, neurovascular remodeling (angiogenesis and neurogenesis), and neuroinflammation. RESULTS Compared with the saline-treated group, exosome-treated rats with TBI showed significant improvement in spatial learning at 34-35 days as measured by the modified Morris water maze test (p < 0.05), and sensorimotor functional recovery (i.e., reduced neurological deficits and foot-fault frequency) was observed at 14-35 days postinjury (p < 0.05). Exosome treatment significantly increased the number of newly generated endothelial cells in the lesion boundary zone and dentate gyrus and significantly increased the number of newly formed immature and mature neurons in the dentate gyrus as well as reducing neuroinflammation. CONCLUSIONS The authors demonstrate for the first time that MSC-generated exosomes effectively improve functional recovery, at least in part, by promoting endogenous angiogenesis and neurogenesis and by reducing inflammation in rats after TBI. Thus, MSC-generated exosomes may provide a novel cell-free therapy for TBI and possibly for other neurological diseases.

Delayed administration of erythropoietin reducing hippocampal cell loss, enhancing angiogenesis and neurogenesis, and improving functional outcome following traumatic brain injury in rats: comparison of treatment with single and triple dose.

OBJECT This efficacy study was designed to investigate traumatic brain injury (TBI) in rats treated with delayed erythropoietin (EPO) administered in a single dose compared with a triple dose. METHODS Young adult male Wistar rats were randomly divided into the following groups: 1) sham group (6 animals); 2) TBI/saline group (6 animals); 3) TBI/EPOx1 group (6 animals); and 4) TBI/EPOx3 group (7 animals). Traumatic brain injury was induced by controlled cortical impact over the left parietal cortex. Erythropoietin (5000 U/kg) or saline was administered intraperitoneally on Day 1 (EPOx1 group) or on Days 1, 2, and 3 (EPOx3 group) postinjury. Neurological function was assessed using a modified neurological severity score, foot-fault, and Morris water maze tests. Animals were killed 35 days after injury and brain sections were stained for immunohistochemistry. RESULTS Compared with the saline treatment, EPO treatment in both the EPOx1 and EPOx3 groups significantly reduced hippocampal cell loss, enhanced angiogenesis and neurogenesis in the injured cortex and hippocampus, and significantly improved neurological functional outcome. The EPOx3 group exhibited significantly improved functional and histological outcomes compared with the EPOx1 group. CONCLUSIONS These data demonstrate that delayed posttraumatic administration of EPO significantly improved histological and long-term functional outcomes in rats after TBI. The triple doses of delayed EPO treatment produced better histological and functional outcomes in rats, although a single dose provided substantial benefits compared with saline treatment.

Therapeutic effects of erythropoietin on histological and functional outcomes following traumatic brain injury in rats are independent of hematocrit

Erythropoietin (EPO) provides neuroprotection and neurorestoration after traumatic brain injury (TBI). The EPO doses used for treatment of TBI significantly increase hematocrit, which may affect the efficacy of EPO therapy for TBI. The aim of this study was to investigate whether normalization of hematocrit would affect EPO efficacy for treatment of TBI. Young adult male Wistar rats were randomly divided into four groups: (1) Sham group (n=6); (2) TBI+ saline group (n=6); (3) TBI+ EPO group (n=6); and (4) TBI+ EPO+ hemodilution group (n=7). TBI was induced by controlled cortical impact over the left parietal cortex. EPO (5,000 U/kg) or saline was administered intraperitoneally at days 1, 2, and 3 postinjury. Neurological function was assessed using a modified neurological severity score (mNSS), footfault and the Morris water maze (MWM) tests. Animals were sacrificed 35 days after injury, and brain sections were stained for immunohistochemistry. Compared to the saline treatment, EPO treatment significantly reduced hippocampal cell loss, enhanced angiogenesis and neurogenesis in the injured cortex and hippocampus, and significantly improved sensorimotor functional outcome (lowered mNSS and foot faults) and spatial learning (MWM test). Normovolemic hemodilution effectively normalized the hematocrit and did not significantly affect the histological and functional outcome of EPO therapy for TBI. These data for the first time demonstrate that increased hematocrit does not affect therapeutic effects of EPO on histological and long-term functional outcomes in rats after TBI and also suggest that neuroprotection and neurorestoration of EPO treatment are independent of hematocrit.

Dose-dependent neurorestorative effects of delayed treatment of traumatic brain injury with recombinant human erythropoietin in rats

OBJECT Delayed (24 hours postinjury) treatment with erythropoietin (EPO) improves functional recovery following experimental traumatic brain injury (TBI). In this study, the authors tested whether therapeutic effects of delayed EPO treatment for TBI are dose dependent in an attempt to establish an optimal dose paradigm for the delayed EPO treatment. METHODS Experimental TBI was performed in anesthetized young adult male Wistar rats using a controlled cortical impact device. Sham animals underwent the same surgical procedure without injury. The animals (8 rats/group) received 3 intraperitoneal injections of EPO (0, 1000, 3000, 5000, or 7000 U/kg body weight, at 24, 48, and 72 hours) after TBI. Sensorimotor and cognitive functions were assessed using a modified neurological severity score and foot fault test, and Morris water maze tests, respectively. Animals were killed 35 days after injury, and the brain sections were stained for immunohistochemical analyses. RESULTS Compared with the saline treatment, EPO treatment at doses from 1000 to 7000 U/kg did not alter lesion volume but significantly reduced hippocampal neuron loss, enhanced angiogenesis and neurogenesis in the injured cortex and hippocampus, and significantly improved sensorimotor function and spatial learning. The animals receiving the medium dose of 5000 U/kg exhibited a significant improvement in histological and functional outcomes compared with the lower or higher EPO dose groups. CONCLUSIONS These data demonstrate that delayed (24 hours postinjury) treatment with EPO provides dose-dependent neurorestoration, which may contribute to improved functional recovery after TBI, implying that application of an optimal dose of EPO is likely to increase successful preclinical and clinical trials for treatment of TBI.

Treatment of traumatic brain injury with thymosin β4 in rats

OBJECT This study was designed to investigate the efficacy of delayed thymosin β(4) (Tβ(4)) treatment of traumatic brain injury (TBI) in rats. METHODS Young adult male Wistar rats were divided into the following groups: 1) sham group (6 rats); 2) TBI + saline group (9 rats); 3) and TBI + Tβ(4) group (10 rats). Traumatic brain injury was induced by controlled cortical impact over the left parietal cortex. Thymosin β(4) (6 mg/kg) or saline was administered intraperitoneally starting at Day 1 and then every 3 days for an additional 4 doses. Neurological function was assessed using a modified neurological severity score (mNSS), foot fault, and Morris water maze tests. Animals were killed 35 days after injury, and brain sections were stained for immunohistochemistry to assess angiogenesis, neurogenesis, and oligodendrogenesis after Tβ(4) treatment. RESULTS Compared with the saline treatment, delayed Tβ(4) treatment did not affect lesion volume but significantly reduced hippocampal cell loss, enhanced angiogenesis and neurogenesis in the injured cortex and hippocampus, increased oligodendrogenesis in the CA3 region, and significantly improved sensorimotor functional recovery and spatial learning. CONCLUSIONS These data for the first time demonstrate that delayed administration of Tβ(4) significantly improves histological and functional outcomes in rats with TBI, indicating that Tβ(4) has considerable therapeutic potential for patients with TBI.

Neuroprotective and neurorestorative effects of thymosin β4 treatment initiated 6 hours after traumatic brain injury in rats.

OBJECT Thymosin β4 (Tβ4) is a regenerative multifunctional peptide. The aim of this study was to test the hypothesis that Tβ4 treatment initiated 6 hours postinjury reduces brain damage and improves functional recovery in rats subjected to traumatic brain injury (TBI). METHODS Traumatic brain injury was induced by controlled cortical impact over the left parietal cortex in young adult male Wistar rats. The rats were randomly divided into the following groups: 1) saline group (n = 7); 2) 6 mg/kg Tβ4 group (n = 8); and 3) 30 mg/kg Tβ4 group (n = 8). Thymosin β4 or saline was administered intraperitoneally starting at 6 hours postinjury and again at 24 and 48 hours. An additional group of 6 animals underwent surgery without TBI (sham-injury group). Sensorimotor function and spatial learning were assessed using the modified Neurological Severity Score and the Morris water maze test, respectively. Animals were euthanized 35 days after injury, and brain sections were processed to assess lesion volume, hippocampal cell loss, cell proliferation, and neurogenesis after Tβ4 treatment. RESULTS Compared with saline administration, Tβ4 treatment initiated 6 hours postinjury significantly improved sensorimotor functional recovery and spatial learning, reduced cortical lesion volume and hippocampal cell loss, and enhanced cell proliferation and neurogenesis in the injured hippocampus. The high dose of Tβ4 showed better beneficial effects compared with the low-dose treatment. CONCLUSIONS Thymosin β4 treatment initiated 6 hours postinjury provides both neuroprotection and neurorestoration after TBI, indicating that Tβ4 has promising therapeutic potential in patients with TBI. These data warrant further investigation of the optimal dose and therapeutic window of Tβ4 treatment for TBI and the associated underlying mechanisms.

Impact of inhibition of erythropoietin treatment-mediated neurogenesis in the dentate gyrus of the hippocampus on restoration of spatial learning after traumatic brain injury.

Our previous study demonstrates that delayed (initiated 24h post injury) erythropoietin (EPO) therapy for traumatic brain injury (TBI) significantly improves spatial learning. In this study, we investigated the impact of inhibition of EPO treatment-mediated neurogenesis on spatial learning after experimental TBI. Young male Wistar rats (318+/-7 g) were subjected to unilateral controlled cortical impact injury. TBI rats received delayed EPO treatment (5000 U/kg in saline) administered intraperitoneally once daily at 1, 2, and 3 days post injury and intracerebroventricular (icv) infusion of either a mitotic inhibitor cytosine-b-D-arabinofuranoside or vehicle (saline) for 14 days. Another 2 groups of TBI rats were treated intraperitoneally with saline and infused icv with either a mitotic inhibitor Ara-C or saline for 14 days. Animals receiving sham operation were infused icv with either Ara-C infusion or saline. Bromodeoxyuridine (BrdU) was administered to label dividing cells. Spatial learning was assessed using a modified Morris water maze test. Animals were sacrificed at 35 days after injury and brain sections stained for immunohistochemical analyses. As compared to the saline treatment, immunohistochemical analysis revealed that delayed EPO treatment significantly increased the number of BrdU-positive cells and new neurons co-stained with BrdU and NeuN (mature neuron marker) in the dentate gyrus in TBI rats. EPO treatment improved spatial learning after TBI. Ara-C infusion significantly abolished neurogenesis and spatial learning recovery after TBI and EPO treatment. Both EPO and Ara-C reduced the number of astrocytes and microglia/macrophages in the dentate gyrus after TBI. Our findings are highly suggestive for an important role of EPO-amplified dentate gyrus neurogenesis as one of the mechanisms underlying EPO therapeutic treatments after TBI, strongly indicating that strategies promoting endogenous neurogenesis may hold an important therapeutic potential for treatment of TBI.

Sprouting of corticospinal tract axons from the contralateral hemisphere into the denervated side of the spinal cord is associated with functional recovery in adult rat after traumatic brain injury and erythropoietin treatment

Erythropoietin (EPO) promotes functional recovery after traumatic brain injury (TBI). This study was designed to investigate whether EPO treatment promotes contralateral corticospinal tract (CST) plasticity in the spinal cord in rats after TBI. Biotinylated dextran amine (BDA) was injected into the right sensorimotor cortex to anterogradely label the CST. TBI was induced by controlled cortical impact over the left parietal cortex immediately after BDA injections. EPO (5000 U/kg) or saline was administered intraperitoneally at Days 1, 2, and 3 post-injury. Neurological function was assessed using a modified neurological severity score (mNSS) and footfault tests. Animals were sacrificed 35 days after injury and brain sections stained for histological analysis. Compared to the saline treatment, EPO treatment significantly improved sensorimotor functional outcome (lower mNSS and reduced footfaults) from Days 7 to 35 post-injury. TBI alone significantly stimulated contralateral CST axon sprouting toward the denervated gray matter of the cervical and lumbar spinal cord; however, EPO treatment further significantly increased the axon sprouting in TBI rats although EPO treatment did not significantly affect axon sprouting in sham animals. The contralesional CST sprouting was highly and positively correlated with sensorimotor recovery after TBI. These data demonstrate that CST fibers originating from the contralesional intact cerebral hemisphere are capable of sprouting into the denervated spinal cord after TBI and EPO treatment, which may at least partially contribute to functional recovery.

Erythropoietin promotes neurovascular remodeling and long-term functional recovery in rats following traumatic brain injury☆

Erythropoietin (EPO) improves functional recovery after traumatic brain injury (TBI). This study was designed to investigate long-term (3 months) effects of EPO on brain remodeling and functional recovery in rats after TBI. Young male Wistar rats were subjected to unilateral controlled cortical impact injury. TBI rats were divided into the following groups: (1) saline group (n=7); (2) EPO-6h group (n=8); and (3) EPO-24h group (n=8). EPO (5000 U/kg in saline) was administered intraperitoneally at 6h, and 1 and 2 days (EPO-6h group) or at 1, 2, and 3 days (EPO-24h group) postinjury. Neurological function was assessed using a modified neurological severity score, footfault and Morris water maze tests. Animals were sacrificed at 3 months after injury and brain sections were stained for immunohistochemical analyses. Compared to the saline, EPO-6h treatment significantly reduced cortical lesion volume, while EPO-24h therapy did not affect the lesion volume (P<0.05). Both the EPO-6h and EPO-24h treatments significantly reduced hippocampal cell loss (P<0.05), promoted angiogenesis (P<0.05) and increased endogenous cellular proliferation (BrdU-positive cells) in the injury boundary zone and hippocampus (P<0.05) compared to saline controls. Significantly enhanced neurogenesis (BrdU/NeuN-positive cells) was seen in the dentate gyrus of both EPO groups compared to the saline group. Both EPO treatments significantly improved long-term sensorimotor and cognitive functional recovery after TBI. In conclusion, the beneficial effects of posttraumatic EPO treatment on injured brain persisted for at least 3 months. The long-term improvement in functional outcome may in part be related to the neurovascular remodeling induced by EPO.

Neuroprotective and neurorestorative effects of thymosin β4 treatment following experimental traumatic brain injury

Traumatic brain injury (TBI) remains a leading cause of mortality and morbidity worldwide. No effective pharmacological treatments are available for TBI because all phase II/III TBI clinical trials have failed. This highlights a compelling need to develop effective treatments for TBI. Endogenous neurorestoration occurs in the brain after TBI, including angiogenesis, neurogenesis, synaptogenesis, oligodendrogenesis, and axonal remodeling, which may be associated with spontaneous functional recovery after TBI. However, the endogenous neurorestoration following TBI is limited. Treatments amplifying these neurorestorative processes may promote functional recovery after TBI. Thymosin beta 4 (Tβ4) is the major G‐actin–sequestering molecule in eukaryotic cells. In addition, Tβ4 has other properties including antiapoptosis and anti‐inflammation, promotion of angiogenesis, wound healing, stem/progenitor cell differentiation, and cell migration and survival, which provide the scientific foundation for the corneal, dermal, and cardiac wound repair multicenter clinical trials. Here, we describe Tβ4 as a neuroprotective and neurorestorative candidate for treatment of TBI.