Adipose Tissue-Derived Mesenchymal Stem Cells (ADMSCs) and ADMSC-Derived Secretome Expedited Wound Healing in a Rodent Model - A Preliminary Study.

INTRODUCTION: The biological role of mesenchymal stem cells (MSCs) in wound healing has been demonstrated. However, there were limited studies on the healing effect of secretome which consists of many biological factors secreted by MSCs. In this study, we aimed to compare the therapeutic effects of secretome with MSCs on facilitating wound healing. METHODS: Green fluorescent protein labelled adipose-derived MSCs (GFP-ADMSCs) or secretome was injected in the full-thickness skin excision model on SD rats. The wound healing process was evaluated by calculating the healing rate and the histological examinations on skin biopsy. The cell viability, proliferation and mobility of the rat dermal fibroblasts were compared after different treatments. The inflammatory response in macrophages was indicated by the level of nitric oxide (NO) and inflammatory cytokines through NO assay and ELISA. RESULTS: On day 5 and day 14, both MSCs and secretome accelerated the wound healing, secretome further enhanced the process. GFP-MSCs were detected 10 days after transplantation. The level of IL-6 and TNF-α in blood was reduced after MSCs and secretome treatments. The expressions of VEGF and PCNA were increased after treatment, higher intensity of VEGF was observed in secretome-injected tissue. The concentrations of total protein and VEGF in secretome were 2.2 ± 0.5 mg/mL and 882.0 ± 72.7 pg/mL, respectively. The cell viability and proliferation of FR were promoted significantly after the treatment. The scratch test showed that secretome accelerated the wound healing speed. Secretome reduced the metabolism of macrophages remarkably, but it did not decrease the level of macrophage-secreted NO. The expression of the pro-inflammatory cytokines (IL-6, MCP-1 and TNF-α) was downregulated significantly. CONCLUSION: Our study indicated both MSCs and MSCs-derived secretome enhanced the wound healing process in early phase. Secretome further promoted the healing effects through promoting the fibroblast proliferation and migration and suppressing the inflammatory response.

Topical application of adipose tissue-derived mesenchymal stem cells (ADMSCs) reduced cerebral edema in experimental traumatic brain injury (TBI)-a preliminary study.

BACKGROUND: Our previous studies showed that topical application of mesenchymal stem cells (MSCs) improved functional recovery in rat traumatic brain injury (TBI) model, and hypoxic precondition further enhanced the therapeutic effects of MSCs. There was no previous study on the attenuation of cerebral edema by MSCs. We investigated whether topical application of normoxic and hypoxic MSCs could reduce cerebral edema in an experimental TBI model. METHODS: Two million normoxic (N = 24) and hypoxic (N = 24) MSCs were applied topically to exposed the cerebral cortex in a controlled cortical impact (CCI) model. The MSCs were fixed in position with fibrin glue. No treatment was given to control animals (TBI only: n = 24). After surgery, four animals in each group were sacrificed daily (day 1 to day 6) for edema evaluation. Normal animals without TBI were used as reference (n = 4). The expressions of GFAP, AQP4, and MMP9 were also investigated by immunofluorescence staining and RT-PCR at day 3. RESULTS: The edema peaked within 3 days after TBI. Compared with the control, hypoxic MSCs reduced brain water content significantly (p < 0.05). Both hypoxic and normoxic MSCs downregulated the expression of MMP9 and normalized AQP4 distribution to astrocyte end feet. CONCLUSION: Our preliminary study showed that topical application of hypoxic MSCs suppressed both vasogenic and cytotoxic edema formation.

The Time Course of Cognitive Deficits in Experimental Subarachnoid Hemorrhage.

Subarachnoid hemorrhage (SAH) is a devastating stroke type. Approximately 50% of survivors suffer from the permanent disability, caused by the cognitive deficits. To enrich the pre-clinical research on the neurological deficits after SAH, we investigate the temporal cognitive deficits and the longitudinal course of cognitive recovery in endovascular perforation SAH murine model. The SAH mice show reproducible body weakness and headache-symbolized moaning symptoms, which is closed to clinical patients. SAH mice exhibit significantly impaired cognitive function in domains of learning ability, short-term and long-term memory. The cognitive deficits occur mostly in the early phase and recover gradually till day 10 after SAH. The systematical assessments of cognitive function after experimental aneurysmal SAH elucidate the time course of cognitive deficits and provide the time window of potential interventions.

Topically applied adipose-derived mesenchymal stem cell treatment in experimental focal cerebral ischemia.

In this study, the neuro-modulation effect of topical mesenchymal stem cells (MSCs) was tested in a rodent middle carotid artery occlusion (MCAO) model. Twenty-four hours after MCAO, craniotomy was made and 0.8 × 106 GFP-MSCs were topically applied to the exposed parietal cortex. The MSCs were fixed in position by a thin layer of fibrin glue (N = 30). In the control group, saline were topically applied to the ipsilateral parietal cortex (N = 30). Three days after topical application, few GFP-positive cells were found in the ischemic penumbra. They expressed GFAP and NeuN. Topical MSCs triggered microglial activation, astrocytosis and cellular proliferation at day 3. The recovery of neurological functions were significantly enhanced as determined in Rotarod test and Morris Water Maze test with smaller infarct volume. PCR array showed that expressions of ten genes of neurogenesis were altered in the penumbra region (fold change > 1.25, p < 0.05) in MSCs group: Apoe, Ascl1, Efnb1, Mef2c, Nog, A100a6 and B2m were up-regulated; Pax2, Pax3 and Th were down-regulated. In conclusion, topical application provided a direct and effective transplant method for the delivery of MSCs to the surface of ipsilateral cerebral cortex and the topical MSCs could improve the neurological function from cerebral ischemia resulting from a major cerebral artery occlusion in a rodent experimental model.

The Dynamics of Microglial Polarization Reveal the Resident Neuroinflammatory Responses After Subarachnoid Hemorrhage.

Neuroinflammation plays a critical role in the pathogenesis of subarachnoid hemorrhage (SAH). Microglia, as the resident immune cells, orchestrate neuroinflammation distinctly in neurological diseases with different polarization statuses. However, microglial polarizations in the neuroinflammatory responses after SAH are not fully understood. In this study, we investigated the dynamics of microglial reaction in an endovascular perforated SAH model. By using the Cx3cr1GFP/GFP Ccr2RFP/RFP transgenic mice, we found that the reactive immune cells were largely from resident microglia pool rather than infiltrating macrophages. Immunostaining and real-time PCR were employed to analyze the temporal microglial polarization and the resulting inflammatory responses. Our results showed that microglia accumulated immediately after SAH with a centrifugal spreading through the Cortex Adjacent to the Perforated Site (CAPS) to the remote motor cortex. Microglia polarized dynamically from M1 to M2 phenotype along with the morphological transformation from ramified to amoeboid shapes. The ramified microglia demonstrated the M1 property, which suggested the function-related microglial polarization occurred prior to morphological transformation after SAH. Bipolar-shaped microglia appeared as the intermediate and transitional status with the capacity of bidirectional polarization. The microglial polarization status is distinct in molecular inflammatory responses; M1-related pro-inflammation was predominant in the early phase and subsequently transited to the M2-related anti-inflammation. The systematic characterization of the dynamics of microglial polarization in this study contributes to the understanding of the origin of neuroinflammatory responses after SAH and provides key foundation for further investigations to develop target treatment.

The neuroprotection of hypoxic adipose tissue-derived mesenchymal stem cells in experimental traumatic brain injury.

Traumatic brain injury is one of the leading causes of mortality and morbidity worldwide. At present there is no effective treatment. Previous studies have demonstrated that topical application of adipose tissue-derived mesenchymal stem cells can improve functional recovery in experimental traumatic brain injury. In this study, we evaluated whether hypoxic preconditioned mesenchymal stem cells could enhance the recovery from traumatic brain injury. Traumatic brain injury was induced with an electromagnetically controlled cortical impact device. Two million mesenchymal stem cells derived from the adipose tissue of transgenic green fluorescent protein Sprague-Dawley rats were cultured under either hypoxic (2.5% O2 for 18 hours) (N = 30) or normoxic (18% O2) (N = 30) conditions, then topically applied to the exposed cerebral cortex within 1 hour after traumatic brain injury. A thin layer of fibrin was used to fix the cells in position. No treatment was given to the animals with traumatic brain injury (N = 30). Animals that underwent craniectomy without traumatic brain injury were treated as the sham group (N = 15). Neurological functions were evaluated with water maze, Roto-rod and gait analysis. Animals were sacrificed at days 3, 7, and 14 for microscopic examinations and real-time polymerase chain reaction analysis. The rats treated with hypoxic mesenchymal stem cells showed the greatest improvement in neurological function recovery. More green fluorescent protein-positive cells were found in the injured brain parenchyma treated with hypoxic mesenchymal stem cells that co-expressed glial fibrillary acidic protein, Nestin, and NeuN. Moreover, there was early astrocytosis triggered by the infiltration of more glial fibrillary acidic protein-positive cells and microgliosis was suppressed with fewer ionized calcium binding adapter molecule 1-positive cells in the penumbra region of hypoxic mesenchymal stem cells group at day 3. Compared with normoxic mesenchymal stem cells and traumatic brain injury only groups, there was significantly (p < 0.05) less neuronal death in both the hippocampus and penumbral regions in sections treated with hypoxic mesenchymal stem cells as determined by Cresyl violet and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling staining respectively. The expression of pro-inflammatory genes (interleukin 6, interleukin 1a, interleukin 1b, tumor necrosis factor α) was upregulated and apoptotic gene (Caspase-3) expression was suppressed at day 3. Anti-inflammatory (interleukin 10) and anti-apoptotic (BCL2 associated agonist of cell death) gene expression was upregulated at days 7 and 14. Our study showed that a hypoxic precondition enhanced the beneficial effects of mesenchymal stem cells on neurological recovery after traumatic brain injury.

Neuroprotective effect of Da Chuanxiong Formula against cognitive and motor deficits in a rat controlled cortical impact model of traumatic brain injury.

ETHNOPHARMACOLOGICAL RELEVANCE: Da Chuanxiong Formula (DCXF) is one of the famous herb pairs that contains dried rhizomes of Ligusticum chuanxiong Hort. and Gastrodia elata Bl. in the mass ratio of 4:1. This classic representative traditional Chinese medicine has been widely used to treat brain diseases like headache and migraine caused by blood stasis and wind pathogen. However, the therapeutic effect of DCXF on traumatic brain injury (TBI) has not been reported yet. AIM OF STUDY: The present study was performed to investigate the neuroprotective effects of DCXF and its underlying mechanisms in the controlled cortical impact (CCI)-induced TBI rat model. MATERIALS AND METHODS: Male Sprague-Dawley rats were divided into four groups: Sham, TBI control, 1X DCXF (520.6 mg/kg) and 5X DCXF (2603.0 mg/kg). Two treatment groups (1X and 5X DCXF) were intragastrically administered daily for 7 days before CCI-induced TBI and then DCXF treatments were continued post-TBI until the animal behavioral tests, including Morris water maze test, acceleration rotarod motor test and CatWalk quantitative gait analysis test, were done. The brain water content and blood brain barrier (BBB) integrity were measured by wet-dry weight method and Evans blue method, respectively. The number of neuron cells, neural stem cells (NSCs), GFAP positive cells (astrocyte) as well as Iba-1 positive cells (microglia) were determined by histology and immunohistochemistry. RESULTS: Treatment with DCXF significantly improved the learning ability and memory retention in Morris water maze test, and remarkably enhanced motor performances in acceleration rotarod motor test and catwalk quantitative gait analysis test after TBI. Moreover, DCXF treatment was able to reduce BBB permeability, brain edema, microglia and astrocyte activation, improve the proliferation of NSCs and decrease neurons loss in the brain with TBI. CONCLUSIONS: The present study demonstrated that DCXF treatment could decrease BBB leakage and brain edema, reduce neuron loss, microglia and astrocyte activation, and increase NSCs proliferation, which may contribute to the cognitive and motor protection of DCXF in the TBI rats. It is the first time to provide potentially underlying mechanisms of the neuroprotective effect of DCXF on TBI-induced brain damage and functional outcomes.

Secretome from hypoxia-conditioned adipose-derived mesenchymal stem cells promotes the healing of gastric mucosal injury in a rodent model.

Studies have indicated that the definitive engraftment and transdifferentiation potential of stem cells do not seem crucial for its property of tissue repair. Our previous study showed that transplantation of adipose-derived mesenchymal stem cells (ADMSCs) enhanced the healing of sutured gastric perforation. This study aimed to investigate the paracrine role of ADMSCs in the experimental gastric mucosal injury. Normoxia-conditioned medium (Nor CM) and hypoxia (HPO) CM were obtained after culturing ADMSCs in 20% O2 and 5% O2 for 48h. Cell migration, proliferation, viability, and angiogenesis in vitro were significantly enhanced upon incubation with CM, especially the HPO CM. Experiments in vivo using a rodent model of gastric ulcer demonstrated that HPO CM treatment significantly accelerated wound healing by suppressing inflammation and promoting neovascularization and re-epithelization. Meanwhile, the infusion of HPO CM activated the COX2-PGE2 axis both in vitro and in vivo. And the upregulation of COX2 was further dependent on the activation of ErK1/2-MAPK pathway. In addition, vascular endothelial growth factor, tissue inhibitors of metalloproteinases-1, and chemokine (C-C motif) ligand 20 (CCL-20) were analyzed as being highly abundant factors secreted by ADMSCs under hypoxic condition. Notably, the blockade of CCL-20 abrogated the HPO CM-induced COX2 signaling in the primary gastric mucosal epithelial cells, while incubation with recombinant CCL-20 increased the expression of COX2. In conclusion, the secretome from hypoxia-conditioned ADMSCs facilitates the repair of gastric mucosal injury through the enhancement of angiogenesis and re-epithelization, as well as the activation of COX2-PGE2 axis with a paracrine activity involving CCL-20 factor.

Topical Application of Mesenchymal Stromal Cells Ameliorated Liver Parenchyma Damage After Ischemia-Reperfusion Injury in an Animal Model.

BACKGROUND: Ischemia-reperfusion injury (IRI) is commonly encountered after liver surgery. This study evaluated the hepatoprotective effects of topically applied adipose-derived mesenchymal stromal cells (ADMSCs) on hepatic IRI in a rat model. METHODS: ADMSCs from transgenic green fluorescent protein Sprague-Dawley rats were topically applied to the liver surface of Sprague-Dawley rats after hepatic IRI and fixed in position by fibrin glue (group A, n = 24). An equivalent amount of ADMSCs were administered through the portal (group B, n = 24) or tail vein (group C, n = 24). In the control group (group D, n = 20), no treatment was given to the IRI liver. RESULTS: All the rats in group A and group D survived. Within 2 days after hepatic IRI, only 50% of rats survived in group B, and ADMSCs were detected in thromboemboli within large vessels. 62.5% of the rats died in group C because most of the ADMSCs were trapped in the lungs. ADMSCs migrated across the liver capsule and homed to the injured liver parenchyma 3 days after topical application in group A. The homed ADMSCs expressed hepatocyte nuclear factor-4α and hepatocyte nuclear factor-1. Compared with group D, the rate of hepatic regeneration in group A was enhanced with less inflammation, smaller necrotic areas, and improved liver function. Proinflammatory cytokines IL-6, IL-21, and CD70 were significantly downregulated in group A by 6.3-, 2.7-, and 12.7-fold, respectively (P < 0.05). The neurogenic locus NOTCH homolog protein pathway was activated in the topical ADMSCs. CONCLUSIONS: Topically applied adipose-derived mesenchymal stromal cells demonstrated hepatoprotective effects on hepatic IRI in an animal model.

Cryostored autologous skull bone for cranioplasty? A study on cranial bone flaps' viability and microbial contamination after deep-frozen storage at -80°C.

Craniectomy is a life-saving procedure. Subsequent cranioplasty with autologous skull bone has a bone resorption rate from 4% to 22.8% and an infection rate from 3.3% to 26%. There are concerns with their viability and the potential microbial contamination as they were explanted for a long period of time. Eighteen cranial bone flaps stored at Prince of Wales Hospital Skull Bone Bank during the period from June 2011 to March 2016 were identified. Ethics approval was obtained. Bone chips and deep bone swabs were collected for osteoblast culture and microbial culture. Skull Bone Bank was kept at -80°C under strict aseptic technique during the study period. The storage period ranged from 4months to 55months. For the osteoblast culture, all eighteen bone flaps had no viable osteoblast growth. For the bacterial culture, five had positive bacteria growth (27.8%). Three were Pasteurella multocida and two were Methicillin-resistant Staphylococcus aureus. The mean duration of storage of the infected bone flap was 32.9months (±15.1months) versus 19.9months (±17.9months) of those bone flaps with no bacterial growth (p=0.1716). The mean size of the infected versus non-infected bone flaps was 117.7cm2 (±44.96cm2) versus 76.8cm2 (±50.24cm2) respectively (p=0.1318). Although in this study statistical significance was not reached, it was postulated that infected bone flaps tended to be larger in size and had a longer duration of storage. In conclusion, cryostored skull bone flaps beyond four months showed no viable osteoblasts. Bacterial contamination rate of bone flaps was 27.8% in this study.

A traditional Chinese formula composed of Chuanxiong Rhizoma and Gastrodiae Rhizoma (Da Chuanxiong Formula) suppresses inflammatory response in LPS -induced RAW 264.7 cells through inhibition of NF-κB pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Da Chuanxiong Formula (DCXF) which origins from Jin Dynasty is a famous classical 2-herb Chinese medicinal prescription. It is composed of dried rhizomes of Ligusticum chuanxiong (Chuanxiong Rhizoma, CR) and Gastrodia elata (Gastrodiae Rhizoma, GR) at the ratio of 4:1 (w/w). It has been used to treat headache which is caused by wind pathogen and blood stasis for thousands of years in China. AIM OF STUDY: The present study was performed to investigate the anti-inflammatory effect of DCXF and elucidate its underlying molecular mechanisms using LPS-stimulated RAW 264.7 cells. MATERIALS AND METHODS: The anti-inflammatory effect of DCXF was evaluated using LPS-stimulated RAW 264.7 cells. Generation of nitric oxide (NO) and prostaglandin E2 (PGE2) were measured by the Griess colorimetric method and enzyme-linked immunosorbent assay (ELISA), respectively. The gene expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were detected by reverse transcription-polymerase chain reaction (RT-PCR). Furthermore, the effect of DCXF on NF-κB activation was measured by western blot assay. RESULTS: Treatment with DCXF significantly suppressed the productions of NO and PGE2 through inhibitions of iNOS and COX-2 expressions in LPS-stimulated RAW 264.7 cells. DCXF significantly decreased IκBα phosphorylation, inhibited p65 expression and reduced p-p65 level. These results suggested the anti-inflammatory effect of DCXF was associated with the reduction of inflammatory mediators through inhibition of NF-κB pathway. CONCLUSIONS: These results indicated that DCXF inhibited inflammation in LPS-stimulated RAW 264.7 cells through inactivation of NF-κB pathway.

The aqueous extract of rhizome of Gastrodia elata Blume attenuates locomotor defect and inflammation after traumatic brain injury in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Traumatic brain injury (TBI) has an incident rate of 200-300 people per 100,000 annually in the developed countries. TBI has relatively high incidence at an early age and may cause long-term physical disability. Patients suffered from severe TBI would have motor and neuropsychological malfunctions, affecting their daily activities. Traditionally, Gastrodia elata Blume is a Chinese Medicines which was used for the head diseases, while their efficiency on reducing brain damage was still largely unknown. In the present study, we aimed to examine the effect of water extract of G. elata Blume (GE) against TBI and elucidate its underlying mechanism. MATERIALS AND METHODS: Sprague-Dawley rats were treated with GE for 7 days, immediately after controlled cortical impact-induced TBI. Impaired neurobehavioral functioning was measured on day 3 and 6 after TBI. Histology of TBI was examined to assess the extent of inflammation, and the expressions of pro-inflammatory cytokines were examined by immunofluorescence study on day 7. RESULTS: GE treatment significantly improved the impaired locomotor functions induced by TBI. GE treatment reduced inflammation and gliosis in the penumbral area. The increase in brain levels of pro-inflammatory cytokines interleukin-6 and tumor necrosis factor-alpha observed in non-GE treated TBI rats were also reversed. CONCLUSIONS: GE treatment attenuated the locomotor deficit caused by TBI. The anti-inflammatory activity might be mediated by inhibition of pro-inflammatory cytokines responses in the TBI-brain.

Transplantation of mesenchymal stem cells to the brain by topical application in an experimental traumatic brain injury model.

Mesenchymal stem cells (MSCs) have been shown in various animal models to be capable of neurorepair and neuroprotection. To carry out a therapeutic function, MSCs must be delivered to the target organ. MSCs are administered to patients via systemic infusion, which has many drawbacks, including a low engraftment rate and the migration of MSCs to non-target organs. However, other approaches such as direct intracerebral injection of MSCs might cause cerebral bleeding. In this study, a traumatic brain injury (TBI) was induced over the right parietal cerebral cortex in Sprague Dawley rats, and green fluorescent protein (GFP)-expressing MSCs (GFP-MSCs), together with a thin layer of fibrin, were applied to the external surface of the contralateral side 2 days later. Within 5 days of topical application, the GFP-MSCs had migrated from the site of application on the cortical surface, through the white matter, and had emerged at the cortical surface of the TBI site on the contralateral cerebral hemisphere, apparently following axons along the corpus callosum. In sham-injured control animals, the topically applied GFP-MSCs proliferated superficially on the cortex at the site of application, and no GFP-MSCs were found at the contralateral cortical surface. In all instances, GFP-MSCs were not detected in other organs of either the test or the control animals. Our study demonstrated that MSCs topically applied to the brain surface can migrate to a TBI site.

Traumatic Brain Injury in the Elderly: Is it as Bad as we Think?

Traumatic brain injury in elderly patients is a neglected global disease burden. The main cause is fall, followed by motor vehicle accidents. This review article summarizes different aspects of geriatric traumatic brain injury, including epidemiology, pathology, and effects of comorbidities and pre-injury medications such as antiplatelets and anticoagulants. Functional outcome with or without surgical intervention, cognitive outcome, and psychiatric complications are discussed. Animal models are also reviewed in attempt to explain the relationship of aging and outcome, together with advances in stem cell research. Though elderly people in general did fare worse after traumatic brain injury, certain "younger elderly" people, aged 65-75 years, could have a comparable outcome to younger adults after minor to moderate head injury.