The Role Of Wharton’s Jelly And (-)-epicatechin In Neuroprotection And Neurogenesis After Traumatic Brain Injury

Traumatic brain injury(TBI) causes extensive neurologic disability and mortality for individuals worldwide. Because of the superficial position of hippocampus, it will be easier disrupted after TBI. Despite progress in diagnosis, neurosurgical care, and neurologic functional recovery for TBI patients, no effective therapeutic treatment is currently available. An understanding of the pathophysiologic mechanisms of TBI and effective treatments that can repair the damaged brain and improve function are urgently needed. In our research, we used Wharton’s Jelly and(-)-Epicatechin respectively to treat TBI for finding a good method to treat TBI.Transplantation of stem cells as one of the effective treatment to treat TBI is widely used. Of the various stem cell types, human umbilical cord mesenchymal stem cells(h UC-MSCs) have many advantages. They display strong self-renewal and differentiation abilities; can be obtained from a wide variety of sources; are easy to collect, culture, and transport; have high reproductive activity; cause no risk of allograft rejection; and pose no ethical controversy. Therefore, h UC-MSCs are one of the most promising types of seed cells for treating TBI Umbilical cord matrix, also known as Wharton’s jelly(WJ), the mucoprotein around human umbilical cord blood vessels composes WJ, which contains abundant hyaluronic acid and collagen. Because of its anti-extrusion and anti-stretching properties, WJ not only supports cell growth, but also acts as a tissue scaffold to produce h UC-MSCs. Therefore, we choose WJ tissue to treat TBI and got good treatment effect.We and others have shown previously that the flavanol(-)-epicatechin(EC), can penetrate the blood-brain barrier(BBB) after intravenous or oral administration and produce a positive effect on cognition and vascular function. EC is thought to offer these benefits by activating the nuclear factor erythroid 2-related factor 2(Nrf2) signaling pathway. By binding to antioxidant response elements(AREs), Nrf2 increases production of proteins involved in reduction of inflammatory damage, oxidative stress, and accumulation of toxic metabolites, as well as proteins that control cell redox state during oxidative stress. In our previous research, after EC treated in the ICH model, EC activated Nrf2-ARE pathway to play the role of neuroprotective. In this research, we used EC to treat CCI model and found the neuroprotective and neurogenesis effect of EC.Part 1. Wharton’s jelly transplantation improves neurologic function in a rat model of traumatic brain injuryObjective Construct the modified weight drop TBI model, testify the neuroprotective effect of Wharton’s Jelly.Method We tested the efficacy of WJ tissue transplantation in a weight drop model of TBI in rats. WJ tissue was cultured and transplanted into the injury site 24 h after TBI. The modified neurologic severity score, body weight, brain edema, and lesion volume were evaluated at various time points after TBI. Cognitive behavior was assessed by the novel object recognition test and the Morris water maze test. Expression of brain-derived neurotrophic factor(BDNF) in the perilesional brain area was measured at day 14 after TBI.Results 1. The identification of h UC-MSCs Flow cytometry analysis showed that h UC-MSCs expressed high levels of matrix marker CD44 and integrin marker CD29 but did not express hematopoietic lineage markers(CD133, CD34, and CD45) or HLA-DR(MHCII). 2. Successfully constructed the TBI model During this study, the mortality was 3.33%(1/33) in the vehicle group and 3.33%(1/33) in the WJ tissue-treated group. 3. WJ tissue treatment decrease lesion volume after TBI On day 28 after TBI, by HE staining, brain lesions were smaller in rats treated with WJ tissue(12.10±1.28 mm3) than in those treated with vehicle(16.00±1.14 mm3 p<0.05). No obvious lesion was observed in the sham-operated rats. 4 WJ tissue treatment increased the body weight after TBI Both vehicle- and WJ tissue-treated rats lost body weight from base line on days 1 and 3 after TBI but began to gain weight on day 7. Rats in the WJ tissue-treated group had gained more body weight than vehicle-treated rats by day28(p<0.05). 5. WJ tissue treatment reduced brain water content after TBI Brain water content on day 3after TBI was significantly greater in the vehicle-treated TBI group than in the sham group. WJ tissue treatment significantly reduced brain water content compared to that in the vehicle-treated group(n=6 rats/group, p<0.05). 6. WJ tissue treatment promoted the motor function after TBI We found no significant difference in m NSS between vehicle- and WJ tissue-treated groups on days 1, 3, 7, and 14 after TBI; however on days 21 and 28, m NSS scores were significantly lower in WJ tissue-treated rats than in vehicle-treated rats(p<0.05). 7. WJ tissue treatment improves cognitive function Rats that underwent TBI exhibited cognitive impairment on day 28 after TBI. In the novel object recognition test, sham rats spent more time exploring the novel object than the old object(p<0.05), but vehicle-treated TBI rats explored novel and old objects for similar lengths of time(p>0.05). However, after WJ tissue treatment, TBI rats spent significantly more time exploring the novel object than the old object(p<0.05). Additionally, the discrimination index was significantly higher in the sham group and in the WJ tissue-treated TBI group than in the vehicle-treated TBI group(p<0.05). Rats in the vehicle-treated group spent less time in the correct quadrant of the Morris water maze than did rats in the sham group on days 26–28 after TBI( p<0.05). The mean percentage of time spent in the correct quadrant was higher in the WJ tissue-treated TBI group than in the vehicle-treated TBI group on days 27 and 28(p<0.05). In addition, the latency to find the platform was significantly longer in the vehicle-treated TBI group than in the sham group from day 24 to 28(n=6 rats/group, p<0.05); however, WJ tissue treatment reduced the time required for rats to find the platform on days 27 and 28(p<0.05). 8. WJ tissue treatment increases BDNF protein and m RNA expression We examined BDNF protein and m RNA expression in the perilesional cortex and subcortical regions on day 14 after TBI. Vehicle-treated TBI rats tended to have higher expression levels of BDNF protein and m RNA than sham-operated rats did, but the difference was not significant(p>0.05); however, BDNF protein and m RNA were significantly higher in the WJ tissue-treated TBI rats than in the vehicle-treated TBI rats on day 14 after treatment(p<0.05). 9. WJ tissue treatment increases the number of MAP2-positive neurons in the perilesional areaWe found that the number of MAP2-positive neurons was increased in the perilesional cortex and subcortex of the WJ tissue-treated group compared with that of the vehicle-treated group. Additionally, the WJ tissue-treated group had more neurons with normal morphology than did the vehicle-treated group on day 14 after TBI(p<0.05).Part 2. Neuroprotection and neurogenesis of(-)-epicatechin after traumatic brain injuryObjective To testify the neuroprotective and neurogenesis effect of(-)-epicatechin(EC) after TBI by Nrf2-ARE pathway.Method We used the controlled cortical impact model to mimic TBI. EC was administered orally at 3 h after TBI and then every 24 h for either 3 or 7 days. We evaluated lesion volume, brain edema, white matter injury, neurologic deficits, cognitive and emotional behaviors, reactive oxygen species(ROS), and a variety of injury-related protein markers. Nrf2 knockout mice were used to determine the role of the Nrf2 signaling pathway in EC treatment. We used Ed U and Ki 67 staining to detect the neurogenesis in SGZ.Results 1. EC treatment reduces short-term lesion volume and neurologic deficit after TBITo identify the optimal dosage of EC for TBI treatment, we administered three doses of EC(5, 15, or 45 mg/kg) after TBI. Cresyl violet staining at 3 days after TBI showed that the lesions were significantly smaller in the 15 mg/kg EC group than in the vehicle group(p<0.05).The neurologic deficit score was significantly lower in the 15 mg/kg group on days 1–3 after TBI. In addition, the wire hanging time was longer in the EC 15 mg/kg group than in the vehicle group on days 1, 2, and 3 post-TBI. In the forelimb placing test, 15 mg/ kg EC increased the placing reflex on days 1 to 3 post-TBI. In the rotarod test, EC at 15 mg increased the latency to fall on days 1, 2, and 3 after TBI compared with that in the vehicle-treated group. Based on the results of these tests, we found that 15 mg/kg was the most efficacious dosage. Therefore, we used this dose in all subsequent studies.2. EC decreases lesion volume and improves neurologic function in the longterm, cell death and neuronal degeneration, increases cell survival and improves brain water content after TBI EC(15 mg/kg) decreased lesion volume compared to that in the vehicle group at 28 days after TBI. EC also improved the neurologic deficit score and results of the forelimb-placing test, the wire-hanging test, and the rotarod test compared with those of the vehicle group(p<0.05). In the NOR test for cognitive ability, the discrimination index decreased from 78.67±3.30% to 55.20±2.55% after TBI, but EC significantly reversed this decrease to 64.43±2.08%. Similarly, TBI caused a decrease in the sucrose preference index compared to the vehicle-treated TBI group. However, EC significantly reversed the effect of TBI on sucrose preference to 67.02±2.17%. In the forced swim test and tail suspension test, the immobility time of the vehicle-treated group was significantly increased after TBI, whereas that of the EC group was decreased compared with that of the vehicle-treated at 28 days after TBI. As EC significantly attenuated brain injury and improved the results of the behavior tests, we further investigated whether this treatment rescued cell death. At 3 days after TBI, EC decreased the number of PI-positive cells compared with that in the vehicle group. Additionally, the EC group had fewer FJB-positive degenerating neurons in the peri-injury area than did the vehicle group and a greater number of surviving neurons(p<0.05). At 3 days post-TBI, brain water content of the ipsilateral hemisphere was significantly lower in the EC-treated group than in the vehicle-treated group(n=6/group, p<0.05).3. EC decreases white matter injury on day 28 post-TBIOn day 28 after TBI, we used Luxol fast blue and MBP staining to label normal myelin in brain sections. Both staining procedures showed that the percentage of area with normal myelin was higher in the EC-treated group than in the vehicle-treated group(p<0.05).4. EC alleviates TBI-induced reactive oxygen species(ROS) production and MMP-9 enzyme activityAfter mice were injected with HEt, ROS were observed as red fluorescence signal around the lesion at 1 day post-TBI. However, the EC-treated mice exhibited less ROS production than did the vehicle-treated mice. We used gelatin gel zymography to detect MMP-2 and MMP-9 enzyme activities. The results showed that EC decreased MMP-9 activity by 51% compared with that in the vehicle-treated group(p<0.05).5. EC reduces Keap-1 expression while increasing Nrf2 nuclear accumulationNext we used Western blot analysis to determine whether EC reduces Keap-1 expression and promotes Nrf2 nuclear translocation. Keap-1 expression was significantly lower in the vehicle-treated TBI group than in the sham group( p<0.05). EC further decreased Keap-1 protein expression compared with that in the vehicle group at 3 days post-TBI. EC did not alter Nrf2 expression in the cytosol(n=6/group, p>0.05), but it did increase nuclear Nrf2 expression compared with that in the vehicle-treated group(p<0.05) at 3 days post-TBI.6. EC increases SOD1 and NQO1 expression and reduces HO-1 expression and iron deposition without reducing the brain hemoglobinAt 3 days after TBI, we examined the expression of transcripts downstream of Nrf2. Expression levels of SOD1 and NQO1 were increased after TBI(p<0.05) compared with those in the sham group and were further increased by EC(p<0.05) compared with levels in the vehicle-treated group. In contrast, expression level of HO-1, the key downstream effector of Nrf2, was upregulated after TBI but was reduced by approximately two-fold after EC treatment(p<0.05).We found that all mice had intracerebral hemorrhage after TBI. Therefore, we measured brain hemoglobin at day 3 after TBI but observed no difference between the vehicle-treated and EC-treated TBI groups(p>0.05). Because HO-1 is the main enzyme to metabolize heme(hemoglobin degradation products) to free iron, we examined ferric iron deposition at 3 days after TBI. EC significantly reduced the number of iron-positive cells compared with that in the vehicle-treated group(p<0.05).7. EC increases neurogenesis in SGZ 3 days after TBI, the Ed U and Ki 67 positive cells in EC treated group are more than in Vehicle treated group(p<0.05).8. EC does not decrease lesion volume or promote neurologic function in Nrf2 KO mice after TBITo verify whether EC protects the brain from injury via the Nrf2 signaling pathway, we subjected Nrf2 KO mice to the TBI model. At 28 days post-TBI, lesion volume in the vehicle-treated and EC-treated Nrf2 KO mice did not differ significantly(p>0.05). However, EC decreased the neurologic deficit score at 3, 7, 14, and 28 days after TBI compared with that in the vehicle-treated group(p<0.05). In the forelimb placing test and the wire-hanging test, we observed no significant difference between the vehicle- and EC-treated groups at any time point(p>0.05). In the rotarod test, the latency to fall was longer in the EC-treated group than in the vehicle treated group on day 3 post-TBI, but not at other time points( p>0.05). At 28 days post-TBI,there was no significant difference between the vehicle- and EC-treated groups in the discrimination index of the NOR test or in the sucrose preference index(p>0.05), although these parameters decreased in the vehicle group compared with those in the sham group(p<0.05). Furthermore, EC did not provide any benefit in the TST or FST. Immobility time in both the TST and FST increased after TBI in the vehicle group(p<0.05) and was not significantly reduced by EC treatment(p>0.05).Conclusion: Wharton’s Jelly protects the TBI brain by increasing the expression of BDNF and the neurons near injury area to promote the motor function and the cognition function.EC protects the TBI brain by activating the Nrf2 pathway, inhibiting HO-1 protein expression, and reducing iron deposition. The latter two effects could represent an Nrf2-independent mechanism in this model of TBI. EC promotes the neurogenesis in the hippocamupus after TBI.