Li-Hua Zhou

Single sevoflurane exposure decreases neuronal nitric oxide synthase levels in the hippocampus of developing rats

BACKGROUND The use of general anaesthetics in young children and infants has raised concerns regarding the adverse effects of these drugs on brain development. Sevoflurane might have harmful effects on the developing brain; however, these effects have not been well investigated. METHODS Postnatal day 7 (P7) Sprague-Dawley rats were continuously exposed to 2.3% sevoflurane for 6 h. We used the Fox battery test and Morris water maze (MWM) to examine subsequent neurobehavioural performance. Cleaved caspase-3 and neuronal nitric oxide synthase (nNOS) were quantified by immunoblotting, and the Nissl staining was used to observe the histopathological changes in the hippocampus. RESULTS A single 6 h sevoflurane exposure at P7 rats resulted in increased cleaved caspase-3 expression and decreased nNOS levels in the hippocampus, and induced the loss of pyramidal neurones in the CA1 and CA3 subfields of the hippocampus at P7-8. These changes were accompanied by temporal retardation of sensorimotor reflexes. However, neither the Fox battery test at P1-21 nor the MWM test at P28-32 showed differences between the air- and sevoflurane-treated groups. CONCLUSIONS Although early exposure to sevoflurane increases activated caspase-3 expression and neuronal loss and decreases nNOS in the neonatal hippocampus, it does not affect subsequent neurobehavioural performances in juvenile rats.

Antisense oligos to neuronal nitric oxide synthase aggravate motoneuron death induced by spinal root avulsion in adult rat

The present study used nitric oxide synthase (nNOS) antisense oligos (nNOS AS-ODN) to assess the role of nNOS in motoneuron death induced by spinal root avulsion. A right seventh cervical (C7) spinal root avulsion was performed on adult male Sprague-Dawley rats. Two weeks later, FITC-labeled random oligos (FITC-R-ODN), nNOS AS-ODN, R-ODN or TE buffer was applied to the lesioned side of the C7 spinal segment and refreshed every 3 days. FITC-R-ODN was first detected inside the injured motoneurons at 10 h, accumulated to a maximum by 24 h and faded out from 72 h. Following avulsion, nNOS AS-ODN decreased the number of nNOS-positive motoneurons in the lesioned segment compared either with buffer (P < 0.001 at 15 days, 3 and 4 weeks post-injury) or with R-ODN control (P = 0.002 at 15 days, P < 0.001 at 3 and 4 weeks post-injury). Interestingly, nNOS AS-ODN also decreased the number of surviving motoneurons compared either with buffer (P = 0.005 at 15 days, P < 0.001 at 3 or 4 weeks) or with R-ODN control (P < 0.001 at 3 or 4 weeks). Meanwhile, there were no significant differences between R-ODN and buffer control either in the number of nNOS-positive motoneurons (P = 0.245 at 15 days, P = 0.089 at 3 weeks and P = 0.162 at 4 weeks) or in the number of surviving motoneurons (P = 0.426 at 15 days, P = 0.321 at 3 weeks or P = 0.344 at 4 weeks). These findings indicate that nNOS AS-ODN, applied from 2 weeks after avulsion, aggravates the motoneuron death due to root avulsion by specifically down-regulating nNOS gene expression and that the expression of nNOS in adult spinal motoneurons in response to root avulsion may play a beneficial role in the survival of injured neurons.

The diversity of nNOS gene expression in avulsion-injured spinal motoneurons among laboratory rodents

Rats, mice, and hamsters are commonly used laboratory rodents in the experimental modeling strategies of avulsion-induced motoneuron degenerations. It is necessary to study the species-specific gene expression in spinal cord in response to avulsion. We carried out the brachial roots avulsion in all animals and compared the survival and nNOS gene expression in injured motoneurons and spinal segments among the three species. The results showed that avulsion-induced loss of motoneurons was greatest in mice than that in hamsters or rats. Avulsion induced decrease of nNOS mRNA level in injured spinal segments in all three species, but greater in amplitude in rats and mice than that in hamsters. However, the de novo nNOS protein expression in injured motoneurons was higher in rats than in hamsters, none in mice. This study strongly suggests that the species diversity of nNOS gene must be considered when estimating the role of nNOS in motoneuron degenerations.

EGb761 protects motoneurons against avulsion-induced oxidative stress in rats

Background Root avulsion of the brachial plexus causes an oxidative stress reaction in the spinal cord and induces dramatic spinal motoneuron death, while EGb761 is a natural free radical cleaning agent. This study was designed to investigate the protective effects of intraperitoneally injected EGb761 against neural damage following brachial root avulsion. Methods The effect of EGb761 on avulsion-induced motoneuron injury was studied in 26 total groups of (n) rats, treated as follows. Animals in singular number groups received EGb761(50 mg/kg.d) and those in complex number groups received normal saline solution (i.p.), serving as controls. Groups 1-8 were used for the determination of nitric oxide (NO) levels in the serum and injured spinal cord at the 5 d, 2 w, 4 w, and 6 w time points. Groups 9-16 were used for determination of constitutive nitric oxide synthase (cNOS) and inducible nitric oxide synthase (iNOS) levels in injured spinal cord at the 5 d, 2 w, 4 w, and 6 w time points. Groups 17-26 were used for determination of the number of neuronal nitric oxide synthase (nNOS)-positive and surviving motoneurons in injured C7 ventral horn at the 5 d, 2 w, 4 w, 6 w and 8 w time points. Results Compared to control groups, the EGb761 treatment group not only had significant decreased levels of NO in serum at 2 w and 6 w after avulsion, but also had reduced levels of NO specifically in the spinal cord at 2 w, 4 w and 6 w. The cNOS activity in the spinal cord was also significant decreased at 2 w and 4 w, while the iNOS activity in injured C6-T1 spinal segments was reduced at 2 w, 4 w and 6 w. All together, the percentages of NADPH-d positive motoneurons in an injured C7 segment were down-regulated and the number of surviving motoneurons in injured C7 ventral horn was increased at 2 w, 4 w, 6 w and 8 w in treated versus untreated animals. Conclusions Intraperitoneal administration of EGb761 after root avulsion of the brachial plexus exerted protective effects by decreasing the level of NO in spinal cord and serum and the activity of cNOS and iNOS, easing the delayed motoneurons death. EGb761 should be considered in the treatment of brachial plexus nerve injuries.

Lithium enhances survival and regrowth of spinal motoneurons after ventral root avulsion

BackgroundDuring the clinical treatment of the brachial plexus root avulsion (BPRA), reimplantation surgery can not completely repair the motor function of the hand because the axonal growth velocity of the spinal motoneurons (MNs) is too slow to re-innervate the intrinsic hand muscles before muscle atrophy. Here, we investigated whether lithium can enhance the regenerative capacity of the spinal MNs in a rat model of BPRA.ResultsThe avulsion and immediate reimplantation of the C7 and C8 ventral roots were performed and followed with daily intraperitoneal administration of a therapeutic concentrationof LiCl. After a 20 week long-term rehabilitation, the motor function recovery of the injured forepaw was studied by a grasping test. The survival and regeneration of MNs were checked by choline acetyltransferase (ChAT) immunofluorescence and by Fluoro-Gold (FG) retrograde labeling through the median and ulnar nerves of the ventral horn MNs. The number and diameter of the nerve fibers in the median nerve were assessed by toluidine blue staining. Our results showed that lithium plus reimplantation therapy resulted in a significantly higher grasping strength of the digits of the injured forepaw. Lithium plus reimplantation allowed 45.1% ± 8.11% of ChAT-positive MNs to survive the injury and increased the number and diameter of nerve fibers in the median nerve. The number of FG-labeled regenerative MNs was significantly elevated in all of the reimplantation animals. Our present data proved that lithium can enhance the regenerative capacity of spinal MNs.ConclusionsThese results suggest that immediate administration of lithium could be used to assist reimplantation surgery in repairing BPRA injuries in clinical treatment.

Activation of phospholipase-Cγ and protein kinase C signal pathways helps the survival of spinal motoneurons injured by root avulsion

J. Neurochem. (2012) 121, 362–372.

Neuroprotective effect of the traditional Chinese herbal formula Tongxinluo： a PET imaging study in rats

Tongxinluo has been widely used in China for the treatment of acute stroke and for neuroprotection. However, there are few positron emission tomography (PET) studies on the neuroprotective effect of Tongxinluo on cerebral ischemia/reperfusion in small animals. In the present study, Tongxinluo superfine powder suspension or its vehicle was administered intragastrically to rats for 5 successive days before middle cerebral artery occlusion. 18F-fluorodeoxyglucose (FDG) small animal PET imaging showed that at 1 and 2 weeks after cerebral ischemia/reperfusion, glucose metabolism in the ischemic area was greater in rats that had received Tongxinluo than in those that had received the vehicle. Nissl staining showed that 2 weeks after cerebral ischemia/reperfusion, there was less neuronal loss in the prefrontal cortex in Tongxinluo-treated rats than in controls. In addition, Tongxinluo-treated animals showed better neurologic function and lower cerebral infarct volume than rats that received the vehicle. These findings suggest that Tongxinluo exhibits neuroprotective effects in cerebral ischemia/reperfusion injury and demonstrates that 18F-FDG small animal PET imaging is a useful tool with which to study the molecular pharmacology of traditional Chinese medicine.

Sevoflurane-induced down-regulation of hippocampal oxytocin and arginine vasopressin impairs juvenile social behavioral abilities.

Cumulative evidence indicates that early childhood anesthesia can alter a child’s future behavioral performance. Animal researchers have found that sevoflurane, the most commonly used anesthetic for children, can produce damage in the neonatal brains of rodents. To further investigate this phenomenon, we focused on the influence of sevoflurane anesthesia on the development of juvenile social behavioral abilities and the pro-social proteins oxytocin (OT) and arginine vasopressin (AVP) in the neonatal hippocampus. A single 6-h sevoflurane exposure for postnatal day 5 mice resulted in decreased OT and AVP messenger RNA (mRNA) and protein levels in the hippocampus. OT and AVP proteins became sparsely distributed in the dorsal hippocampus after the exposure to sevoflurane. Compared with the air-treated group, mice in the sevoflurane-treated group showed signs of impairment in social recognition memory formation and social discrimination ability. Sevoflurane anesthesia reduces OT and AVP activities in the neonatal hippocampus and impairs social recognition memory formation and social discrimination ability in juvenile mice.

Neuronal nitric oxide synthase, as a downstream signaling molecule of c‑jun, regulates the survival of differentiated PC12 cells

The high expression of c-jun and neuronal nitric oxide synthase (nNOS) generally occurs in neurons following the generation of various animal models of central neuronal diseases. However, the mechanism between them in neuronal disease remains to be elucidated. Our previous studies demonstrated that the expression of c‑jun always occurs prior to expression of nNOS in motoneuron injuries and suppression of c‑jun expression by c‑jun siRNA decreased nNOS expression in differentiated PC12 cells. The present study aimed to examine whether there was an association of up and downstream regulation or crosstalk between c‑jun and nNOS in neurons. Using a culture of differentiated PC12 cells in vitro, the expression of nNOS and c-jun in cells was investigated by immunofluorescence. The nNOS inhibitor 7‑nitroindazole (7‑NI) was used in differentiated PC12 cells to downregulate the expression of nNOS. The optimal concentration of 7‑NI on the viability and survival of cultured differentiated PC12 cells was selected using a 3‑(4,5-dimethylthiazol-2-yl)‑2,5-diphenyltetrazolium assay and the effects of 7‑NI on the activity of constitutive nitric oxide synthase (cNOS) in differentiated PC12 cells were determined using a NOS Activity Detection kit. The effects of 7‑NI on the gene expression of nNOS and c‑jun were detected by western blot analysis. The results from the immunofluorescence demonstrated that the c‑jun and nNOS protein were constantly expressed in PC12 cells. The cell viability of differentiated PC12 cells were significantly inhibited by treatment with 200 and 400 µmol/l 7‑NI, and the expression levels of the nNOS protein were significantly inhibited by treatment with 200 µmol/l 7‑NI. However, 7‑NI had no significant effect on the protein expression level of c‑jun and the total activities of cNOS. Based on our previous studies, which revealed that the nNOS gene was a downstream signaling molecule of the JNK/c‑jun signaling pathway in cultured neurons, the expression of nNOS downstream was able to be regulated by c‑jun which was the upstream molecule. Therefore, these results indicated that the association between them involved up and downregulation instead of crosstalk.

Involvement of phospholipase C-γ in the pro-survival role of glial cell line-derived neurotrophic factor in developing motoneurons in rat spinal cords

The glial cell line-derived neurotrophic factor (GDNF) has been proven to be the most powerful neurotrophic factor in neuronal development. However, it remains uncertain as to which intracellular signaling pathway interacting with GDNF is invovlved in motoneuron (MN) development. In this study, we investigated whether phosphoinositide phospholipase C-γ (PLC-γ) is involved in GDNF-promoted MN development. The primary spinal MNs from 12- to 14-day-old embryos of Sprague-Dawley rats were cultured and survival was sustained by GDNF. A specific inhibitor of PLC-γ, 1-[6-((17b-3-methoxyestra-1,3,5(10)-trien-17-yl) amino)hexyl]-1H-pyrrole-2,5-dione (U73122), was used to block the pro-survival effect of GDNF. Our results showed that MN-like cells appeared at 72 h after initial implantation and were sustained for a period of up to seven days under GDNF treatment. These cultured MNs expressed neuron-specific enolase, SMI-32, 75-kDa low-affinity neurotrophic receptor and choline acetyltransferase. The survival rate of the cultured MNs at 24 h was significantly lower in the GDNF + U73122-treated group (31.87±2.17%), compared either with that of the GDNF- (81.38±1.13%) or GDNF + DMSO (79.39±1.22%)-treated groups. The present data suggest that PLC-γ may be one of the intracellular signals that play a role in the survival-promoting effects of GDNF in developing spinal MNs.