Primary dissociated neuronal cultures are widely used research tools to investigate of pathological mechanisms and to treat various central and peripheral nervous system problems including trauma and degenerative neuronal diseases. We introduced a protocol that utilizes hippocampal and cortical neurons from embryonic day 17 or 18 mice. We applied appropriate markers (GAP-43 and synaptophysin) to investigate whether neurite outgrowth and synaptogenesis can be distinguished at a particular period of time. GAP-43 was found along the neural processes in a typical granular pattern, and its expression increased proportionally as neurites lengthened during the early in vitro period. Unlike GAP-43, granular immunoreactive patterns of synaptophysin along the neurites were clearly found from day 2 in vitro with relatively high immunoreactive levels. Expression of synaptic markers from cortical neurons reached peak level earlier than that of hippocampal neurons, although neurite outgrowths of hippocampal neurons were faster than those of cortical neurons. The amount of peak synaptic markers expressed was also higher in cortical neurons than that in hippocampal neurons. These results strongly suggest the usefulness of primary cultured neurons from mice embryos for synaptic function and plasticity studies, because of their clear and typical patterns of morphology that establish synapses. Results from this study also suggest the proper amount of time in vitro according to neuronal types (cortical or hippocampal) when utilized in experiments related with synaptogenesis or synaptic activities.
Animals ; Embryonic Structures ; GAP-43 Protein ; Mice ; Neurites ; Neurons ; Peripheral Nervous System ; Plastics ; Synapses ; Synaptophysin

The genetically engineered mice require special husbandry care and are mainly housed in Individually Ventilated Cage (IVC) systems and Static Micro Isolator Cages (SMIC) to minimize the risk for spreading undesirable microorganisms. However, the static micro isolation cage housing like SMIC are being replaced with IVC systems in many facilities due to a number of benefits like a higher density housing in limited space, better protection from biohazards and allergens and decreased work load due to decreased frequency of cage changing required in this system. The purpose of this study was to examine the reproductive performance of genetically engineered mice housed in individually ventilated cages (IVC) and Static Micro Isolator Cages (SMIC). When the B6C3-Tg (APPswe, PSEN1dE9) 85Dbo/Mmjax transgenic mice were housed in these two housing systems, the number of litters per dam, number of pups born per dam and number of pups weaned per dam were found to be slightly higher in the IVC as compared to the SMIC but the difference was not significant (P<0.05). In case of Growth Associated Protein 43 (GAP-43) knockout mice, the number of litters born per dam and the number of pups born per dam were marginally higher in the IVC as compared to those housed in SMIC but the difference was not significant (P<0.05). Only the number of pups weaned per dam were found to be significantly higher as compared to those housed in the SMIC system at P<0.05.
Allergens ; Animals ; GAP-43 Protein ; Hazardous Substances ; Housing* ; Mice* ; Mice, Knockout ; Mice, Transgenic

Although growth associated protein-43 (GAP-43) is known to play a significant role in the regulation of axonal growth and the formation of new neuronal connections in the hippocampus, there is only a few studies on the effects of acute stress on GAP-43 mRNA expression in the hippocampus. Moreover, the effects of repeated citalopram treatment on chronic mild stress (CMS)-induced changes in GAP-43 mRNA expression in the hippocampus have not been explored before. To explore this question, male rats were exposed to acute immobilization stress or CMS. Also, citalopram was given prior to stress everyday during CMS procedures. Acute immobilization stress significantly increased GAP-43 mRNA expression in all subfields of the hippocampus, while CMS significantly decreased GAP-43 mRNA expression in the dentate granule cell layer (GCL). Repeated citalopram treatment decreased GAP-43 mRNA expression in the GCL compared with unstressed controls, but this decrease was not further potentiated by CMS exposure. Similar decreases in GAP-43 mRNA expression were observed in CA1, CA3 and CA4 areas of the hippocampus only after repeated citalopram treatment in CMS-exposed rats. This result indicates that GAP-43 mRNA expression in the hippocampus may differently respond to acute and chronic stress, and that repeated citalopram treatment does not change CMS-induced decreases in GAP-43 mRNA expression in the GCL.
Animals ; Axons ; Citalopram ; GAP-43 Protein ; Hippocampus ; Humans ; Immobilization ; Male ; Neurons ; Rats ; RNA, Messenger

OBJECTIVE: To verify the effect of low energy laser irradiation (LELI) on the regeneration of injured sciatic nerve of the rat by showing the functional improvement and the elevated immunoreactivities (IRs) of growth-associated protein 43 (GAP-43). METHOD: Twenty rats, which had standardized compression injuries to the sciatic nerves, received the calculated LELI therapy immediately after the nerve injury and four consecutive days. The functional status was evaluated by sciatic functional index (SFI), and GAP-43-IRs was evaluated by immunohistochemistry and RT-PCR. RESULTS: The SFI was recovered in LELI rats faster than in the control group. Although expression of GAP-43 in the injured sciatic nerve was increased both in the LELI and control groups, the intensities of GAP-43-IRs were much greater in LELI treated group at 1 and 3 weeks after nerve injury. Both SFI and GAP-43-IRs reached the same level at 5 weeks after the nerve injury. CONCLUSION: LELI enhanced the neural regeneration after experimentally induced sciatic nerve injury at the early stage of recovery. Considering the effect of LELI on nerve regeneration was not fully explained until now, this study could suggest the meaningful explanation on the mechanism of LELI effectiveness on neural regeneration.
Animals ; GAP-43 Protein ; Immunohistochemistry ; Nerve Regeneration ; Peripheral Nerve Injuries ; Rats* ; Regeneration* ; Sciatic Nerve*

OBJECTIVE: To observe the effect of moxibustion at "Huantiao" (GB 30) on the expression of growth-associated protein-43 (GAP-43) in the sciatic nerve trunk and ventral horn of spinal cord (L METHODS: A total of 48 healthy male SD rats were randomly divided into a normal group, a sham operation group, a model group and a moxibustion group, 12 rats in each group. The rat model of primary sciatic pain was established by chronic constriction injury (CCI) of the sciatic nerve in the model group and the moxibustion group. On the 8th day of the experiment, moxibustion was adopted at "Huantiao" (GB 30) in the moxibustion group for 5-10 min, once a day for 14 consecutive days. Sciatic nerve function index (SFI) was measured and compared in each group at day 1, 7, 14 and 21. On the 21st day of the experiment, HE staining was used to observe the morphology of ventral horn of rat spinal cord and sciatic nerve trunk. Immunohistochemical method and real-time PCR were used to detect mRNA and protein expressions of GAP-43 in the spinal cord and sciatic nerve trunk of rats. RESULTS: On day 7, 14 and 21, there was no statistical difference in SFI between the sham operation group and the normal group ( CONCLUSION Moxibustion at "Huantiao" (GB 30) could improve the sciatic nerve function in rats with primary sciatica and its mechanism may be related to improving the expression of GAP-43 and enhancing the self-repair ability of the sciatic nerve after injury.
Animals ; Electroacupuncture ; GAP-43 Protein/genetics* ; Male ; Moxibustion ; Rats ; Rats, Sprague-Dawley ; Sciatic Nerve ; Sciatica/therapy* ; Spinal Cord

OBJECTIVETo investigate whether there is neogenesis of myelin sheath and neuron after transplantation of Schwann cells into cerebral hemorrhage lesion.

METHODSSchwann cells were expanded, labeled with BrdU in vitro and transplanted into rat cerebral hemorrhage with blood extracted from femoral artery and then injected into the basal nuclei. Double immunohistochemistry staining and electron microscopy were used to detect the expression of BrdU/MBP and BrdU/GAP-43 and remyelination.

RESULTSBrdU/MBP double positive cells could be seen at 1 week up to 16 weeks after transplantation of Schwann cells. Thin remyelination was observed under electron microscope. GAP-43 positive cells appeared after 12 weeks and were found more in Hippocamp.

CONCLUSIONSGrafted Schwann cells participate in remyelination and promoter nerve restore in rat cerebral hemorrhage.

Animals ; Cerebral Hemorrhage ; metabolism ; therapy ; GAP-43 Protein ; metabolism ; Rats ; Rats, Wistar ; Schwann Cells ; metabolism ; transplantation ; Sciatic Nerve ; cytology ; embryology

The neuronal phosphoprotein GAP-43 has been implicated in neuritogenesis during developmental stages of the nervous system and in regenerative processes and neuronal plasticity in the adult. It was previously reported that the level of increased GAP-43 expression in facial motor neurons following crush injury or nerve resection was paralleled the process of axonal regeneration. Since the optimal timing of repair after facial nerve transection injury has been debated for several decades, the author designed this study for the evaluation of optimal timing of facial nerve repair after injury by using the level of GAP-43 expression in facial motor neurons as a marker of neural regeneration. The increased expression of GAP-43 in facial motor neurons was returned to undetectable level by 4 weeks following the compression injury and by 8 weeks following the nerve transection and immediate end-to-end anastomosis, while it was maintained for at least 16 weeks after the nerve ligation and 1 week delayed anastomosis. This study suggests that the best functional outcomes could be obtained when the transected facial nerve is repaired as early as possible.
Adult ; Animals ; Axons ; Facial Nerve Injuries* ; Facial Nerve* ; GAP-43 Protein* ; Humans ; Immunohistochemistry ; Ligation ; Motor Neurons* ; Nervous System ; Neuronal Plasticity ; Neurons ; Rats* ; Regeneration

Most epileptic patients have commonly suffered from recurrent seizures for many years. These seizures are usually associated with inhibitory synaptic reorganization of the hippocampal region, but it is not known whether cerebellar inhibitory synaptic changes can be induced by seizure activity. We sought to determine the pattern of cerebellar alterations in the cerebellar inhibitory interneurons (basket and stellate cells) and then tested if the alterations are associated with their synaptic transmission at the cerebellar GABAergic synapses between inhibitory interneurons and Purkinje cells after systemic kainic acid administration by immunohistochemistry, western blot analysis, dot blot analysis and confocal microscopy. A dramatic increase of the intensity of GAP-43 immunostaining was obvious in the pinceau structures following KA-induced seizures and the intense GAP-43 immunoreaction involved in high expression of PKC-sigma. The activation of the presynaptic terminal at the cerebellar inhibitory synapse is accompanied with strong GABA immunoreactivity in pinceau region (especially 48 h) after KA-seizures. These results suggest a possibility that KA-seizures increase the release of GABA at the cerebellar inhibitory presynaptic terminal and it would be contribute to the depression of Purkinje cell activity, disinhibition, during the epileptogenesis.
Blotting, Western ; Depression ; gamma-Aminobutyric Acid ; GAP-43 Protein ; Humans ; Immunohistochemistry ; Interneurons* ; Kainic Acid ; Microscopy, Confocal ; Presynaptic Terminals ; Purkinje Cells ; Seizures* ; Synapses* ; Synaptic Transmission

The present study was designed to investigate the role of quercetin on neurite growth in N1E-115 cells and the underlying mechanisms. Quercetin was evaluated for its effects on cell numbers of neurites, neurite length, intracellular cAMP content, and Gap-43 expression in N1E-115 cells in vitro by use of microscopy, LANCE(tm) cAMP 384 kit, and Western blot analysis, respectively. Our results showed that quercetin could increase the neurite length in a concentration-dependent manner, but had no effect on the numbers of cells. Quercetin significantly increased the expression of cellular cAMP in a time- and concentration-dependent manner. The Gap-43 expression was up-regulated in a time-dependent manner. In conclusion, quercetin could promote neurite growth through increasing the intracellular cAMP level and Gap-43 expression.
Cell Line ; Cyclic AMP ; metabolism ; GAP-43 Protein ; metabolism ; Nerve Regeneration ; Neurites ; drug effects ; Plant Extracts ; pharmacology ; Quercetin ; pharmacology ; Signal Transduction

OBJECTIVETo observe time points of the expressions of basic fibroblast growth factor (bFGF), growth associated protein-43 (GAP-43) and neurogenesis after cerebral ischemia/reperfusion in rats and explore its possible mechanism of neurogenesis.

METHODSModels of middle cerebral artery occlusion (MCAO) were established in SD rats which were divided into 3 d, 7 d, 14 d and 28 d groups (n = 6). The neurological severity was evaluated by neurological severity scores (NSS) and scores of motor test (SMT). Neuronal injury in the boundary zone of the infarction area was evaluated by TUNEL and Nissl staining; The expressions of bFGF and GAP-43 and neurogenesis were evaluated by Western blot and 5-bromodeoxyuridine (Brdu) fluorescence staining, respectively.

RESULTSIt showed up neurologic impairment and motor dysfunction after cerebral ischemia/reperfusion in rats at 3 d, the numbers of neuron apoptosis also peaked at 3d, the protein levels of bFGF and GAP-43 were significantly increased in time-dependent manner, peaked at 7 d and then decreased gradually, meanwhile, Brdu and NeuN double fluorescence staining displayed scattered Brdu-and NeuN-positive cells in the boundary zone of the infarction area.

CONCLUSIONThese results suggest that the upregulation of bFGF and GAP-43 may contribute to the neurogenesis after cerebral ischemia/reperfusion.

Animals ; Brain Ischemia ; metabolism ; physiopathology ; Fibroblast Growth Factor 2 ; metabolism ; GAP-43 Protein ; metabolism ; Male ; Neurogenesis ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; metabolism ; physiopathology