The effect of Hes1 upregulation on neurogenesis after traumatic brain injury

Objective To construct and evaluate a mouse model of traumatic brain injury (TBI) that simulates both motor and cognitive impairment. Methods Twenty-four healthy male C57BL/6 mice were randomly divided into a sham group and a TBI group (n=12/group). The TBI model was prepared by referring to the compression injury model with some modifications. The sham group underwent an identical process without mechanical trauma. Motor function was evaluated using the rotarod and beam-walking tasks at 1, 3, 7, 14, 21, 28 days post-injury. Spatial learning and memory capacities were assessed at 28, 29, 30, 31, 32, 33 days post-injury by the Morris Water Maze (MWM) test. Nissl staining was performed to observe pathological changes and immunofluorescence staining to detect the expression of glial fibrillary acidic protein (GFAP) and ionized calcium binding adaptor protein (Iba-1) in the mouse brain on the 34th day after modeling. Results The latency for the TBI group was significantly lower than that for the sham group, and the frequency of slipping off the beam by the right hindlimbs for the TBI group was significantly higher than that for the sham group at 1, 3, 7, 14, 21, 28 days post-injury (P<0.05). The escape latency for the TBI group was significantly longer than that for the sham group in the MWM test at 30, 31 and 32 days after modeling (P<0.05). The times of crossing the platform for the TBI group were significantly less than those for the sham group at day 33 after TBI (P< 0.05). The lesion volume for the sham group was significantly smaller than that for the TBI group [(0.55±0.06)% vs. (16.90±1.14)%, P<0.05]. The numbers of astrocytes in the TBI and sham groups were respectively 101.40±6.18/mm2 and 20.17±1.55/mm2, and the numbers of microglia in the 2 groups were respectively 119.20±6.28/mm2 and 23.58±1.72/mm2, showing statistically significant differences between the 2 groups (P<0.05). Conclusion Since the TBI model we constructed is simple and reproducible with stable motor deficits and cognitive impairments which are consistent with the pathological changes of moderate TBI, it can be used in animal TRI experiments. Key words: Traumatic brain injury; Model, animal; Movement deficit; Cognitive impairment

Evidence for the generation of young neurons out of precursor cells in the adult brain, i.e. adult neurogenesis, exists for at least two brain regions. New nerve cells are generated in the subventricular zone of the olfactory bulb and in the subgranular zone of hippocampal dentate gyrus. Young neurons of the subgranular zone migrate along the rostral migratory stream to the olfactory bulb, where they functionally integrate and contribute to the discrimination of odors. In the hippocampus the function of newly formed granule cells is still a matter of debate, yet it is thought that adult neurogenesis functionally contributes to hippocampal functions.&#13;\nOver the last twenty years of extensive research it became clear that adult hippocampal neurogenesis (AHN) in laboratory rodents can be up-and down regulated by different internal and external stimuli. Physical exercise in a running wheel being among the factors that have been most investigated. Since voluntary exercise not only increases adult neurogenesis in the hippocampus but also beneficially affects learning and memory in laboratory mice and rats, a widespread assumption holds a direct relationship between AHN and cognitive brain health also in higher order species, including humans. However, translating findings in laboratory rodents to the human condition faces difficulties. Enormous differences in basal rates of adult neurogenesis have been reported between mammalian species. The low level of AHN in primates and the complete lack of adult neurogenesis in bat species indicate species-specific differences in adult neurogenesis not only on a regulatory but also on a functional level. For a better understanding of species-specific differences in the regulation of AHN, we investigated basal rates of adult neurogenesis in laboratory mice and closely related wild mouse strains as well as the reaction of AHN to motivationally different running conditions. Testing different wild- and laboratory mice in the same environment allowed the identification of species-specific differences as well as possible domestication effects.&#13;\nBasal rates of adult hippocampal neurogenesis in equally-aged and genetically identical laboratory C57BL/6 mice show individual differences possibly reflecting epigenetic factors. However, the initial level of adult neurogenesis does not influence the response to wheel-exercise. Voluntary physical exercise in laboratory mice always increases AHN but this positive effect cannot be additively stimulated by enhanced running and is even lost as soon as the mice are forced to run. Rewarding the mice for their performance leads to an increase in wheel activity&#13;\nbut does not translate into a corresponding additive increase in adult neurogenesis. Likewise, a more naturalistic situation, in which laboratory mice must run to obtain their daily food does not lead to an increase in cell proliferation and entails only a small increase in the number of young neurons, far below the one in voluntary running mice.&#13;\nWild wood mice (Apodemus sylvaticus) and wild-derived western house mice (Mus musculus domesticus), both close relatives of the common laboratory mouse strains, were tested in the same running situations as laboratory C57BL/6 mice. Besides species-differences in basal neurogenesis rate, we find adult neurogenesis in wild mice remaining relatively constant in response to external influences. None of the factors that normally affect AHN in laboratory animals, such as stress, environmental changes or physical exercise, have an effect on adult neurogenesis in these animals. In wood mice, neither voluntary wheel running nor stress or an impoverished cage environment affect the number of newly generated neurons. House mice also show a stable adult neurogenesis, which shows no significant change after voluntary running or running for food.&#13;\nAdult neurogenesis in the dentate gyrus is thus regulated differently in laboratory and wild mice. However, even in laboratory mice it is not as plastic as initially suggested: laboratory mice, which are tested in a more naturalistic and complex running situation, show rather weak plasticity of AHN, resembling wild mice. Hence, it seems that the regulatory difference in adult neurogenesis between laboratory- and wild mice is, that laboratory animals react to a single stimulus in absence of other inputs. We believe that the constant exposure to different stimuli potentially affecting AHN has led to a natural selection that stabilizes adult neurogenesis in the wild. In contrast, during domestication - including inbreeding - much of the homeostatic capacity in regulating adult neurogenesis might have been lost.&#13;\nTaken together, our data imply that genetic (species-specific differences as well as within-species variation) play an important role in determining basal rates of adult neurogenesis, while motivational-contextual factors modulate the response of AHN to physical exercise, albeit chiefly in domesticated laboratory strains . As such differences appear already between phylogenetically closely related species, extrapolating findings in laboratory mice to distantly related taxonomic groups, such as humans, obviously requires much caution.

Objective To investigate whether mild hypothermia can promote neurogenesis in the dentate gyrus of hippocampus and cognitive function recovery after traumatic brain injury (TBI) through inhibiting apoptosis of hippocampal neurons. Methods A total of 66 healthy adult Sprague-Dawley rats were randomly divided into sham group, TBI group and TBI+ hypothermia group, with 22 rats in each group. The rat TBI model was established using the fluid percussion device. The rats in TBI+ hypothermia group received 4-hour hypothermia therapy immediately after injury, with the target temperature of 33.5℃. Bromodeoxyuridine (BrdU) was injected into the rats' abdominal cavity to label the mitotic cells. The test of Morris water maze was used to evaluate the rats' spatial learning and memory capabilities. Immunofluorescence staining was used to observe the expression levels of BrdU, doublecortin (DCX), neuron specific nuclear protein (NeuN), cysteinyl aspartate specific proteinase 3 (caspase-3) and cleaved caspase-3 expressions in dentate gyrus of hippocampus at 7 days and 28 days after injury. Expressions apoptosis-related proteins including the factor associated suicide (FAS)/factor associated suicide ligand (FASL), B-cell lymphoma-2 (Bcl-2), caspase-3 and cleaved caspase-3 expressions were detected by Western blot assay. Results The water maze tests at 28 days after injury showed that compared with TBI group, the escape latency in TBI+ hypothermia group was significantly shorter [(24.2±5.9)s∶(18±4.1)s], and both the time in the target quadrant and the number of platform crossing were increased significantly [(24.9±6.5)s∶(31.7±5.2)s; (1.9±0.8) times∶(3.5±1.2)times](P<0.05). Compared with the sham group, in TBI group and TBI+ hypothermia group, the BrdU+ new-born cells in the dentate gyrus of hippocampus were significantly increased at 7 days after injury [(9.4±4.1)∶(33.4±3.8); (9.4±4.1)∶(45.8±5.6)], the BrdU+ /DCX+ new-born neurons were increased at 7 days after injury [(2.0±0.6)∶(9.6±1.6); (2.0±0.6)∶(19.2±3.7)], and the BrdU+ /NeuN+ mature neurons were increased at 28 days after injury [(2.6±1.0)∶(17.2±3.9); (2.6±1.0)∶(33.6±9.1)] (P<0.01). TBI group showed more obvious increase than the TBI+ hypothermia group (P<0.01). Moreover, compared with 7 days after injury, the number of BrdU+ cells at 28 days after injury was further increased in TBI+ hypothermia group but decreased in TBI group [(45.8±5.6)∶(58.8±9.2); (33.4±3.8)∶(22.0±3.5)](P<0.05 or <0.01). Compared with the sham group, the caspase-3+ NeuN+ and caspase-3+ NeuN+ apoptotic neurons were significantly increased at 7 days after injury in TBI group [(2.0±0.9)∶(11.6±2.6); (2.6±1.0)∶(10.2±2.9)] (P<0.05). Compared with the TBI group, the cleaved caspase-3+ NeuN+ apoptotic neurons were decreased in TBI+ hypothermia group [(6.6±2.0)∶(11.6±2.6)](P<0.05). Furthermore, compared with the TBI group, mild hypothermia might down-regulate the expression of FAS, FASL, cleaved caspase-3 and caspase-3 and up-regulate the expression of Bcl-2 in the hippocampus [(1.54±0.15) ∶(1.14±0.12); (1.06±0.04)∶(0.80±0.09); (0.84±0.03)∶(0.62±0.08); (0.93±0.06)∶(0.86±0.09); (0.71±0.01)∶(1.58±0.18)](P<0.05). Conclusions Mild hypothermia might inhibit apoptosis of hippocampal neurons through cleaved caspase-3, FAS/FASL and Bcl-2 pathways, thus improving the neurogenesis and maturation of neurons in the dentate gyrus of hippocampus and facilitating cognitive function recovery in rats. It indicates that the function of hypothermia in anti-apoptosis and neurogenesis and maturity of hippocampal neurons may have a potential role in predicting the prognosis of TBI patients. Key words: Hypothermia; Brain injuries; Hippocampus; Neuve regeneration; Apoptosis

Objective To explore the neuroprotective effects of dexmedetomidine (Dex) combined with targeted temperature management (TTM) on brain edema in traumatic brain injury (TBI) mice. Methods A total of 132 male C57BL/6 mice were randomly divided into normal group, sham-operation (Sham) group, traumatic brain injury (TBI) group, targeted temperature management (TTM) group, dexmedetomidine (Dex) group, and combination with TTM and Dex (DT) group, respectively (n=22 per group). The TBI animal model was established by electric controlled cortical impactor (eCCI), and then settled promptly on a mild hypothermic blanket (targeted temperature of (32±1)℃) for 6 h, or intraperitoneal injection of Dex (20 μl/kg) at 0, 2 and 4 h after TBI. Neurological function and spatial learning and memory ability were evaluated by modified neurological severity scores (mNSS) and Morris water maze (MWM), respectively. Then, mice were sacrificed at 24 h after TBI and stained using hematoxylin and eosin (H & E). Additionally, the cerebral edema was evaluated from the water content of the brain tissue using the wet-to-dry weight ratio, and the expression of aquaporin 4 (AQP4) was measured by Western blot assay. Results Compared with the Sham group, TBI mice showed neurologic deficits ((13.2±3.0) vs (0.5±0.7))(P<0.01), spatial learning and memory capacity decline((2.9±1.0) vs (7.4±1.3))( P<0.05), and the brain water content and the expression of AQP4 were increased ((79.81±0.80)% vs (75.98±0.62)%): ((1.60±0.07) vs (0.73±0.03))(all P<0.01). However, Dex or TTM could improve the neurological function(Dex: (10.3±2.8), TTM: (10.0±2.9)), reduce the brain water content(Dex: (78.50±0.40)%, TTM: (78.10±0.45)%), and significantly decrease the expression of AQP4 compared with the TBI group(Dex: (1.40±0.04), TTM: (1.15±0.12)) (all P<0.05), especially that of DT group ((9.2±2.5) , (5.4±1.8) , (77.67±0.30)%, (0.93±0.09)) (all P<0.01). Conclusion These finding suggests that Dex combined with targeted temperature management can reduce cerebral edema and improve neurological outcome in mice after TBI to exert neuroprotection effect, which may be related with the down-regulation of AQP4 protein. Key words: Traumatic brain injury; Dexmedetomidine; Targeted temperature management; Brain edema

Objective To examine the effect of survivin downregulation by adenovirus-mediated RNA interference on apoptosis in hippocampal dentate gyrus and spatial learning and memory of mice with traumatic brain injury (TBI). Methods A total of 112 adult male mice were allocated into 4 groups (28 animals each) according to the random number table. Fluid percussion model of TBI was used in the study. In negative control group, animals were stereotactically injected with control recombinant adenovirus immediately after injury. In survivin knockdown group, the animals were stereotactically injected with shRNA adenovirus immediately after injury. In sham group, animals were subjected to identical surgery without fluid percussion injury. In TBI group, animals were subjected to isolated fluid percussion injury. Real-time fluorescent quantitative polymerase chain reaction (RT-PCR), Western blot analysis and immunofluorescence staining were used to detect expression level of survivin in mouse brain tissue. TUNEL assay was used for measuring apoptotic cells in hippocampal denate gyrus. Morris water maze test was adopted to evaluate mouse learning and memory after surviving downregulation. Results Expression levels of survivin mRNA and protein in survivin knockdown group reduced at days 3 postinjury compared to negative control group (1.21±0.11 vs.4.05±0.30; 0.34±0.08 vs.1.11±0.17, respectively) (P<0.01). Immunofluorescence staining showed survivin-positive cells calculated 267.45±38.41 in survivin knockdown group, lower than 896.56±102.23 in negative control group (P<0.01). TUNEL-positive cells in dentate gyrus of the hippocampus in survivin knockdown group increased at days 3 postinjury compared to negative control group (416.1±29.0 vs. 250.2±20.8) (P<0.01). Morris water maze test confirmed that survivin downexpression did lead to a statistically significant changes in escape latency (P<0.05) and target quadrant (P<0.01). Conclusion Decreased expression of survivin may promote apoptotic cell death and result in a negative role in the recovery of learning and memory function of mice after TBI. Key words: Mice, knockout; Brain injuries; Apoptosis; Memory function

Hes1, a Notch signaling downstream target, regulates adult hippocampal neurogenesis following traumatic brain injury

Secreted Frizzled-Related Protein 3 Regulates Activity-Dependent Adult Hippocampal Neurogenesis

Downregulation of survivin regulates adult hippocampal neurogenesis and apoptosis, and inhibits spatial learning and memory following traumatic brain injury

Hippocampal Adult Neurogenesis Is Maintained by Neil3-Dependent Repair of Oxidative DNA Lesions in Neural Progenitor Cells

Young children who have sustained severe traumatic brain injury (TBI) can suffer from debilitating neurocognitive deficits. Impairment of adult hippocampal neurogenesis is associated with cognitive deficits and depression. Very few studies have investigated the adult hippocampal neurogenesis after pediatric TBI. Here, we evaluated long-term cognition, adult hippocampal neurogenesis, and microglial activation in a rabbit pediatric TBI model. On Post-natal Day 5-7 (P5-7), New Zealand white rabbits from the same litter were randomized into naïve, sham (craniotomy alone), and TBI (controlled cortical impact). Bromodeoxyuridine (BrdU, 50 mg/kg, intraperitoneally) was administered at 1-month post-injury, once/daily for 5 consecutive days. Novel object recognition and spontaneous alternation in T-maze tests were performed at 2 months post-injury to measure the cognitive functions. The animals were euthanized after behavioral tests at 3 months of age to evaluate adult hippocampal neurogenesis and microglial activation. We found that: 1) pediatric TBI caused significant deficits in hippocampal dependent cognitive functions; 2) the survival rates of adult-born neurons at both ipsilateral and contralateral hippocampus significantly decreased in the TBI group; 3) TBI induced ectopic migration of adult-born neurons at the dorsal dentate gyrus in both ipsilateral and contralateral hippocampus; 4) TBI increased astrogenesis in the hilus of the dentate gyrus; and 5) TBI results in abnormal microglial activation. In conclusion, pediatric TBI causes prolonged neuroinflammation and dysregulation of the adult hippocampal neurogenesis through young adulthood, which might be responsible for the cognitive deficits. Protection of adult hippocampal neurogenesis may potentially improve outcomes.

Traumatic brain injury (TBI) is an important cause of death in young adults and children. Till now, the treatment of TBI in the short- and long-term complications is still a challenge. Our previous evidence implied aquaporin 4 (AQP4) and hypoxia inducible factor-1α (HIF-1α) might be potential targets for TBI. In this study, we explored the roles of AQP4 and HIF-1α on brain edema formation, neuronal damage and neurological functional deficits after TBI using the controlled cortical injury (CCI) model. The adult male Sprague Dawley rats were randomly divided into sham and TBI group, the latter group was further divided into neutralized-AQP4 antibody group, 2-methoxyestradiol (2-ME2) group, and their corresponding control, IgG and isotonic saline groups, respectively. Brain edema was examined by water content. Hippocampal neuronal injury was assessed by neuron loss and neuronal skeleton related protein expressions. Spatial learning and memory deficits were evaluated by Morris water maze test and memory-related proteins were detected by western blot. Our data showed that increased AQP4 protein level was closely correlated with severity of brain edema after TBI. Compared with that in the control group, both blockage of AQP4 with neutralized-AQP4 antibody and inhibition of HIF-1α with 2-ME2 for one-time treatment within 30–60 min post TBI significantly ameliorated brain edema on the 1st day post-TBI, and markedly alleviated hippocampal neuron loss and spatial learning and memory deficits on the 21st day post-TBI. In summary, our preliminary study revealed the short-term and long-term benefits of targeting HIF-1α-AQP4 axis after TBI, which may provide new clues for the selection of potential therapeutic targets for TBI in clinical practice.

Objective To establish a stable reproducible repeated traumatic brain injury (TBI) animal model to study the neuron loss after TBI. Methods Sixty-four male SD rats were randomly divided into single TBI control group, the single TBI group, repetitive TBI control group, and repetitive TBI group, and each group was divided into four subgroups: 3 d group, 7 d group, 14 d group and 30 d group, each subgroup of 4 rats. Single TBI group was hit only once after craniotomy, repetitive TBI group hit four times in intervals of 24 h, and the control group was only given anesthesia to remove bone flap. After TBI model was established, the modified neurological severity score (mNSS) was detected at 1st, 3rd, 7th, 14th and 30th day, and Morris water maze test was used to examine spatial learning and memory ability at 30th day. The hematoxylin and eosin (HE) staining was used to observe the brain damage after the injury, and immunofluorescence to test neurons NeuN missing after injury. Results (1) As compared with the single TBI group, the mNSS results showed that the neurobehavioral injury was more serious at different time points after injury (1, 3 d: P=0.013, 7 d: P=0.003, 14 d: P=0.008, 21, 30 d: P=0.000). Morris water maze latency training results showed that the time to reach the target platform was prolonged at the second day after the start of training when compared with single TBI group (P=0.000). The percentage of target quadrant showed that the single injury group [(33.33±6.80)%] was higher than that in the repeated injury group [(16.34±3.76)%] in the same injury time (P=0.029). (2) Immunofluorescence showed that the neurons in the repetitive TBI group around the damaged area and those in the area of hippocampal dentate gyrus were lost seriously over time, more significant than in single TBI group (damaged area: 14 d: P=0.007, 30 d: P=0.003; hippocampal dentate gyrus: 14 d: P=0.009, 30 d: P=0.007). Conclusion As compare with the single TBI group, repetitive TBI leads to severe neurological defect and neuron loss. This repetitive TBI model can provide replicable rat model to study the neuropathology mechanism of neurodegeneration after repetitive brain injury. Key words: Traumatic brain injury; Neurodegenerative disease; Rat; Neuron; Model, animal

Adult hippocampal neurogenesis in Alzheimer's disease: A roadmap to clinical relevance.

Neurogenesis involves generation of new neurons through finely tuned multistep processes, such as neural stem cell (NSC) proliferation, migration, differentiation, and integration into existing neuronal circuitry in the dentate gyrus of the hippocampus and subventricular zone. Adult hippocampal neurogenesis is involved in cognitive functions and altered in various neurodegenerative disorders, including Alzheimer disease (AD). Ethosuximide (ETH), an anticonvulsant drug is used for the treatment of epileptic seizures. However, the effects of ETH on adult hippocampal neurogenesis and the underlying cellular and molecular mechanism(s) are yet unexplored. Herein, we studied the effects of ETH on rat multipotent NSC proliferation and neuronal differentiation and adult hippocampal neurogenesis in an amyloid β (Aβ) toxin-induced rat model of AD-like phenotypes. ETH potently induced NSC proliferation and neuronal differentiation in the hippocampus-derived NSC in vitro. ETH enhanced NSC proliferation and neuronal differentiation and reduced Aβ toxin-mediated toxicity and neurodegeneration, leading to behavioral recovery in the rat AD model. ETH inhibited Aβ-mediated suppression of neurogenic and Akt/Wnt/β-catenin pathway gene expression in the hippocampus. ETH activated the PI3K·Akt and Wnt·β-catenin transduction pathways that are known to be involved in the regulation of neurogenesis. Inhibition of the PI3K·Akt and Wnt·β-catenin pathways effectively blocked the mitogenic and neurogenic effects of ETH. In silico molecular target prediction docking studies suggest that ETH interacts with Akt, Dkk-1, and GSK-3β. Our findings suggest that ETH stimulates NSC proliferation and differentiation in vitro and adult hippocampal neurogenesis via the PI3K·Akt and Wnt·β-catenin signaling.

Objectives Childhood traumatic brain injury (TBI) is a public health issue. Our objectives were to determine the prevalence of permanent pituitary hormone deficiency and to detect the emergence of other pituitary dysfunctions or central precocious puberty several years after severe TBI. Design Follow-up at least 5 years post severe TBI of a prospective longitudinal study. Patients Overall, 66/87 children, who had endocrine evaluation 1 year post severe TBI, were included (24 with pituitary dysfunction 1 year post TBI). Main outcome measures In all children, the pituitary hormones basal levels were assessed at least 5 years post TBI. Growth hormone (GH) stimulation tests were performed 3-4 years post TBI in children with GH deficiency (GHD) 1 year post TBI and in all children with low height velocity (<-1 DS) or low IGF-1 (<-2 DS). Central precocious puberty (CPP) was confirmed by GnRH stimulation test. Results Overall, 61/66 children were followed up 7 (5-10) years post TBI (median; (range)); 17/61 children had GHD 1 year post TBI, and GHD was confirmed in 5/17 patients. For one boy, with normal pituitary function 1 year post TBI, GHD was diagnosed 6.5 years post TBI. 4/61 patients developed CPP, 5.7 (2.4-6.1) years post-TBI. Having a pituitary dysfunction 1 year post TBI was significantly associated with pituitary dysfunction or CPP more than 5 years post TBI. Conclusion Severe TBI in childhood can lead to permanent pituitary dysfunction; GHD and CPP may appear after many years. We recommend systematic hormonal assessment in children 1 year after severe TBI and a prolonged monitoring of growth and pubertal maturation. Recommendations should be elaborated for the families and treating physicians.