Early distribution of18 F-labeled AAV9 vectors in the cerebrospinal fluid after intracerebroventricular or intracisternal magna infusion in non-human primates.

BACKGROUND: The delivery of adeno-associated virus (AAV) vectors via the cerebrospinal fluid (CSF) has emerged as a valuable method for widespread transduction in the central nervous system. Although infusion into the cerebral ventricles is a common protocol in preclinical studies of small animals, the cisterna magna has been recognized as an alternative target for clinical studies because it can be reached in a less invasive manner using an intrathecal catheter via the subarachnoid space from a lumbar puncture. METHODS: We evaluated the early distribution of fluorine-18-labeled AAV9 vectors infused into the lateral ventricle or cisterna magna of four non-human primates using positron emission tomography. The expression of the green fluorescent protein was immunohistochemically determined. RESULTS: In both approaches, the labeled vectors diffused into the broad arachnoid space around the brain stem and cervical spinal cord within 30 min. Both infusion routes efficiently transduced neurons in the cervical spinal cord. CONCLUSIONS: For gene therapy that primarily targets the cervical spinal cord and brainstem, such as amyotrophic lateral sclerosis, cisterna magna infusion would be a feasible and effective administration method.

Global brain delivery of neuroligin 2 gene ameliorates seizures in a mouse model of epilepsy.

BACKGROUND: Despite the increasing availability of effective drugs, around one-third of patients with epilepsy are still resistant to pharmacotherapy. Gene therapy has been suggested as a plausible approach to achieve seizure control, in particular for patients with focal epilepsy. Because seizures develop across wide spans of the brain in many forms of epilepsy, global delivery of the vectors is necessary to tackle such generalized seizures. Neuroligin 2 (NL2) is a postsynaptic cell adhesion molecule that induces or strengthens inhibitory synaptic function by specifically combining with neurexin 1. METHODS: In the present study, we applied an adeno-associated virus (AAV) type 9 vector expressing NL2 to modulate neuronal excitability in broad areas of the brain in epileptic (EL) mice, a model of polygene epilepsy. We administered the AAV vector expressing Flag-tagged NL2 under the synapsin I promoter (AAV-NL2) via cardiac injection 6 weeks after birth. RESULTS: Significant reductions in the duration, strength and frequency of seizure were observed during a 14-week observation period in NL2-treated EL mice compared to untreated or AAV-green fluorescent protein-treated EL mice. No behavioral abnormality was observed in NL2-treated EL mice in an open-field test. Immunohistochemical examination at 14 weeks after AAV-NL2 injection revealed the expression of exogenous NL2 in broad areas of the brain, including the hippocampus and, in these areas, NL2 co-localized with postsynaptic inhibitory molecule gephyrin. CONCLUSIONS: Global brain delivery of NL2 by systemic administration of AAV vector may provide a non-invasive therapeutic approach for generalized epilepsy.

Survivin overexpression via adeno-associated virus vector Rh10 ameliorates ischemic damage after middle cerebral artery occlusion in rats.

Survivin, a member of the inhibitors of apoptosis protein gene family, inhibits the activity of caspase, leading to a halt of the apoptotic process. Our study focused on the neuroprotective effect of survivin after transient middle cerebral artery occlusion (MCAO) with intraparenchymal administration of an adeno-associated virus (AAV) vector. His-tagged survivin was cloned and packaged into the AAV-rh10 vector. Four-week-old Sprague-Dawley rats were injected with 4 × 109  vg of AAV-GFP or AAV-His-survivin into the right striatum and were treated 3 weeks later with transient MCAO for 90 min. Twenty-four hours after MCAO, functional and histological analyses of the rats were performed. The result showed that rats that had been treated with AAV-green fluorescent protein (GFP) and those that had been treated with AAV-His-survivin did not show a significant difference in neurological scores 24 hr after MCAO, however, infarction volume was significantly reduced in the AAV-His-survivin group compared to that in the AAV-GFP group. Although the neutrophil marker myeloperoxidase did not show a significant difference in the ischemic boundary zone, cells positive for active caspase-3 and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling were significantly decreased in the AAV-His-survivin group. In conclusion, survivin overexpression in the ischemic boundary zone induced by using an AAV vector has the potential for amelioration of ischemic damage via an antiapoptotic mechanism.

Gene therapy for Glut1-deficient mouse using an adeno-associated virus vector with the human intrinsic GLUT1 promoter.

BACKGROUND: We generated an adeno-associated virus (AAV) vector in which the human SLC2A1 gene, encoding glucose transporter type 1 (GLUT1), was expressed under the human endogenous GLUT1 promoter (AAV-GLUT1). We examined whether AAV-GLUT1 administration could lead to functional improvement in GLUT1-deficient mice. METHODS: We extrapolated human endogenous GLUT1 promoter sequences from rat minimal Glut1 promoter sequences. We generated a tyrosine-mutant AAV9/3 vector in which human SLC2A1-myc-DDK was expressed under the human GLUT1 promoter (AAV-GLUT1). AAV-GLUT1 was administered to GLUT1-deficient mice (GLUT1+/- mice) via intracerebroventricular injection (1.85 × 1010 vg/mouse or 6.5 × 1010 vg/mouse). We analyzed exogenous GLUT1 mRNA and protein expression in the brain and other major organs. We also examined improvements of cerebral microvasculature, motor function using rota-rod and footprint tests, as well as blood and cerebrospinal fluid (CSF) glucose levels. Additionally, we confirmed exogenous GLUT1 protein distribution in the brain and other organs after intracardiac injection (7.8 × 1011 vg/mouse). RESULTS: Exogenous GLUT1 protein was strongly expressed in the cerebral cortex, hippocampus and thalamus. It was mainly expressed in endothelial cells, and partially expressed in neural cells and oligodendrocytes. Motor function and CSF glucose levels were significantly improved following intracerebroventricular injection. Exogenous GLUT1 expression was not detected in other organs after intracerebroventricular injection of AAV-GLUT1, whereas it was detected in the liver and muscle tissue after intracardiac injection. CONCLUSIONS: Exogenous GLUT1 expression after AAV-GLUT1 injection approximated that of physiological human GLUT1 expression. Local central nervous system administration of AAV-GLUT1 improved CSF glucose levels and motor function of GLUT1-deficient mice and minimized off-target effects.

Adeno-associated Virus-mediated Ezh2 Knockdown Reduced the Increment of Newborn Neurons Induced by Forebrain Ischemia in Gerbil Dentate Gyrus.

It is established that neurogenesis of dentate gyrus is increased after ischemic insult, although the regulatory mechanisms have not yet been elucidated. In this study, we focused on Ezh2 which suppresses gene expression through catalyzing trimethylation of lysine 27 of histone 3. Male gerbils were injected with adeno-associated virus (AAV) carrying shRNA targeting to Ezh2 into right dentate gyrus 2 weeks prior to forebrain ischemia. One week after ischemia, animals were injected with thymidine analogue to label proliferating cells. Three weeks after ischemia, animals were killed for histological analysis. AAV-mediated knockdown of Ezh2 significantly decreased the ischemia-induced increment of proliferating cells, and the proliferated cells after ischemia showed significantly longer migration from subgranular zone (SGZ), compared to the control group. Furthermore, the number of neural stem cells in SGZ significantly decreased after ischemia with Ezh2 knockdown group. Of note, Ezh2 knockdown did not affect the number of proliferating cells or the migration from SGZ in the non-ischemic condition. Our data showed that, specifically after ischemia, Ezh2 knockdown shifted the balance between self-renewal and differentiation toward differentiation in adult dentate gyrus.

Higher Transduction Efficiency of AAV5 to Neural Stem Cells and Immature Neurons in Gerbil Dentate Gyrus Compared to AAV2 and rh10.

The safety and high efficiency of adeno-associated virus (AAV) vectors has facilitated their wide-scale use to deliver therapeutic genes for experimental and clinical purposes in diseases affecting the central nervous system (CNS). AAV1, 2, 5, 8, 9, and rh10 are the most commonly used serotypes for CNS applications. Most AAVs are known to transduce genes predominantly into neurons. However, the precise tropism of AAVs in the dentate gyrus (DG), the region where persistent neurogenesis occurs in the adult brain, is not fully understood. We stereotaxically injected 1.5 × 1010 viral genomes of AAV2, 5, or rh10 carrying green fluorescent protein (GFP) into the right side of gerbil hippocampus, and performed immunofluorescent analysis using differentiation stage-specific markers 1 week after injection. We found that AAV5 showed a significantly larger number of double-positive cells for GFP and Sox2 in the DG, compared with the AAV2 and rh10 groups. On the contrary, AAVrh10 presented a substantially larger number of double-positive cells for GFP and NeuN in the DG, compared with AAV2 and AAV5. Our findings indicated that AAV5 showed high transduction efficiency to neural stem cells and precursor cells, whereas AAVrh10 showed much higher efficiency to mature neurons in the DG.

Deficiency of neural recognition molecule NB-2 affects the development of glutamatergic auditory pathways from the ventral cochlear nucleus to the superior olivary complex in mouse.

Neural recognition molecule NB-2/contactin 5 is expressed transiently during the first postnatal week in glutamatergic neurons of the central auditory system. Here, we investigated the effect of NB-2 deficiency on the auditory brainstem in mouse. While almost all principal neurons are wrapped with the calyces of Held in the medial nucleus of the trapezoid body (MNTB) in wild type, 8% of principal neurons in NB-2 knockout (KO) mice lack the calyces of Held at postnatal day (P) 6. At P10 and P15, apoptotic principal neurons were detected in NB-2 KO mice, but not in wild type. Apoptotic cells were also increased in the ventral cochlear nucleus (VCN) of NB-2 KO mice, which contains bushy neurons projecting to the MNTB and the lateral superior olive (LSO). At the age of 1 month, the number of principal neurons in the MNTB and of glutamatergic synapses in the LSO was reduced in NB-2 KO mice. Finally, interpeak latencies for auditory brainstem response waves II-III and III-IV were significantly increased in NB-2 KO mice. Together, these findings suggest that NB-2 deficiency causes a deficit in synapse formation and then induces apoptosis in MNTB and VCN neurons, affecting auditory brainstem function.

Protection against aminoglycoside-induced ototoxicity by regulated AAV vector-mediated GDNF gene transfer into the cochlea.

Since standard aminoglycoside treatment progressively causes hearing disturbance with hair cell degeneration, systemic use of the drugs is limited. Adeno-associated virus (AAV)-based vectors have been of great interest because they mediate stable transgene expression in a variety of postmitotic cells with minimal toxicity. In this study, we investigated the effects of regulated AAV1-mediated glial cell line-derived neurotrophic factor (GDNF) expression in the cochlea on aminoglycoside-induced damage. AAV1-based vectors encoding GDNF or vectors encoding GDNF with an rtTA2s-S2 Tet-on regulation system were directly microinjected into the rat cochleae through the round window at 5 x 10(10) genome copies/body. Seven days after the virus injection, a dose of 333 mg/kg of kanamycin was subcutaneously given twice daily for 12 consecutive days. GDNF expression in the cochlea was confirmed and successfully modulated by the Tet-on system. Monitoring of the auditory brain stem response revealed an improvement of cochlear function after GDNF transduction over the frequencies tested. Damaged spiral ganglion cells and hair cells were significantly reduced by GDNF expression. Our results suggest that AAV1-mediated expression of GDNF using a regulated expression system in the cochlea is a promising strategy to protect the cochlea from aminoglycoside-induced damage.

Protection Against Aminoglycoside-induced Ototoxicity by Regulated AAV Vector-mediated GDNF Gene Transfer Into the Cochlea.

Since standard aminoglycoside treatment progressively causes hearing disturbance with hair cell degeneration, systemic use of the drugs is limited. Adeno-associated virus (AAV)-based vectors have been of great interest because they mediate stable transgene expression in a variety of postmitotic cells with minimal toxicity. In this study, we investigated the effects of regulated AAV1-mediated glial cell line-derived neurotrophic factor (GDNF) expression in the cochlea on aminoglycoside-induced damage. AAV1-based vectors encoding GDNF or vectors encoding GDNF with an rtTA2s-S2 Tet-on regulation system were directly microinjected into the rat cochleae through the round window at 5 × 1010 genome copies/body. Seven days after the virus injection, a dose of 333 mg/kg of kanamycin was subcutaneously given twice daily for 12 consecutive days. GDNF expression in the cochlea was confirmed and successfully modulated by the Tet-on system. Monitoring of the auditory brain stem response revealed an improvement of cochlear function after GDNF transduction over the frequencies tested. Damaged spiral ganglion cells and hair cells were significantly reduced by GDNF expression. Our results suggest that AAV1-mediated expression of GDNF using a regulated expression system in the cochlea is a promising strategy to protect the cochlea from aminoglycoside-induced damage.

Hippocampal GAD67 Transduction Using rAAV8 Regulates Epileptogenesis in EL Mice.

Gene therapy has been employed as a therapeutic approach for intractable focal epilepsies. Considering the potential of focal GABAergic neuromodulation in regulating epileptogenesis, the GABA-producing enzyme, γ-aminobutyric acid decarboxylase 67 (GAD67), is highly suitable for epilepsy therapy. The EL/Suz (EL) mouse is a model of multifactorial temporal lobe epilepsy. In the present study, we examined focal gene transduction in epileptic EL mice using recombinant adeno-associated virus serotype 8 (rAAV8) expressing human GAD67 to enhance GABA-mediated neural inhibition. Eight-week-old mice were bilaterally injected with rAAV8-GFP or rAAV8-GAD67 in the hippocampal CA3 region. After four weeks, the GAD67-transduced EL mice, but not the rAAV-GFP-treated EL mice, exhibited a significant reduction in seizure generation. The GAD67-mediated depression became stable after 14 weeks. The excitability of the CA3 region was markedly reduced in the GAD67-transduced EL mice, consistent with the results of the Ca2+ imaging using hippocampal slices. In addition, downregulation of c-Fos expression was observed in GAD67-transduced hippocampi. Our findings showed that rAAV8-GAD67 induced significant changes in the GABAergic system in the EL hippocampus. Thus, rAAV8-mediated GAD67 gene transfer is a promising therapeutic strategy for the treatment of epilepsies.

Antagonism of sphingosine 1-phosphate receptor-2 enhances migration of neural progenitor cells toward an area of brain.

BACKGROUND AND PURPOSE: We have previously shown that the sphingosine 1-phosphate (S1P)/S1P receptor-1 (S1P(1)R) axis contributes to the migration of transplanted neural progenitor cells (NPCs) toward areas of spinal cord injury. In the current study, we examined a strategy to increase endogenous NPC migration toward the injured central nervous system to modify S1PR. METHODS: S1P concentration in the ischemic brain was measured in a mouse thrombosis model of the middle cerebral artery. NPC migration in vitro was assessed by a Boyden chamber assay. Endogenous NPC migration toward the insult was evaluated after ventricular administration of the S1P(2)R antagonist JTE-013. RESULTS: The concentration of S1P in the brain was increased after ischemia and was maximal 14 days after the insult. The increase in S1P in the infarcted brain was primarily caused by accumulation of microglia at the insult. Mouse NPCs mainly expressed S1P(1)R and S1P(2)R as S1PRs, and S1P significantly induced the migration of NPCs in vitro through activation of S1P(1)R. However, an S1P(1)R agonist failed to have any synergistic effect on S1P-mediated NPC migration, whereas pharmacologic or genetic inhibition of S1P(2)R by JTE-013 or short hairpin RNA expression enhanced S1P-mediated NPC migration but did not affect proliferation and differentiation. Interestingly, administration of JTE-013 into a brain ventricle significantly enhanced endogenous NPC migration toward the area of ischemia. CONCLUSIONS: Our findings suggest that S1P is a chemoattractant for NPCs released from an infarcted area and regulation of S1P(2)R function further enhances the migration of NPCs toward a brain infarction.

Efficient transduction of adeno-associated virus vectors into gerbil hippocampus with an appropriate combination of viral capsids and promoters.

Adeno-associated virus (AAV) is an ideal vector for gene transduction into the central nervous system because of its safety and efficiency. While it is currently widely used for clinical trials and is expected to become more widespread, the appropriate combination of viral serotypes and promoters have not been fully investigated. In this study, we compared the transduced gene expression of AAVrh10 to AAV5 in gerbil hippocampus using three different promoters, including cytomegalovirus (CMV), chicken ß-actin promoter with the CMV immediate-early enhancer (CAG), and the Synapsin 1 (Syn1) promoter. Four-week-old male gerbils underwent stereotaxic injection with 1 × 1010 viral genome of AAV carrying green fluorescent protein (GFP). Quantification of the GFP-positive areas 3 weeks after injection showed that AAVrh10-CMV and AAVrh10-CAG were the most efficient (p < 0.001, compared with the control) and AAVrh10-Syn1 and AAV5-CMV were the next most efficient (p < 0.05, compared with the control). On the other hand, AAV5-Syn1 showed little expression, which was only observed at the injected site. In conclusion, we should note that some combinations of viral capsids and promoters can result in failure of gene delivery, while most of them will work appropriately in the transgene expression in the brain.

Gene therapy for a mouse model of glucose transporter-1 deficiency syndrome.

OBJECTIVE: We generated an adeno-associated virus (AAV) vector in which the human SLC2A1 gene was expressed under the synapsin I promoter (AAV-hSLC2A1) and examined if AAV-hSLC2A1 administration can lead to functional improvement in GLUT1-deficient mice. METHODS: AAV-hSLC2A1 was injected into heterozygous knock-out murine Glut1 (GLUT1+/-) mice intraperitoneally (systemic; 1.85 × 1011 vg/mouse) or intra-cerebroventricularly (local; 1.85 × 1010 vg/mouse). We analyzed GLUT1 mRNA and protein expression, motor function using rota-rod and footprint tests, and blood and cerebrospinal fluid (CSF) glucose levels. RESULTS: Vector-derived RNA was detected in the cerebrum for both injection routes. In the intra-cerebroventricular injection group, exogenous GLUT1 protein was strongly expressed in the cerebral cortex and hippocampus near the injection site. In the intraperitoneal injection group, exogenous GLUT1 protein was mildly expressed in neural cells throughout the entire central nervous system. The motor function test and CSF/blood glucose ratio were significantly improved following intra-cerebroventricular injection. CONCLUSIONS: AAV-hSLC2A1 administration produced exogenous GLUT1 in neural cells and improved CSF glucose levels and motor function of heterozygous knock-out murine Glut1 mice.

Large-scale production of recombinant viruses by use of a large culture vessel with active gassing.

Adenovirus and adeno-associated virus (AAV) vectors are increasingly used for gene transduction experiments. However, to produce a sufficient amount of these vectors for in vivo experiments requires large-capacity tissue culture facilities, which may not be practical in limited laboratory space. We describe here a large-scale method to produce adenovirus and AAV vectors with an active gassing system that uses large culture vessels to process labor- and cost-effective infection or transfection in a closed system. Development of this system was based on the infection or transfection of 293 cells on a large scale, using a large culture vessel with a surface area of 6320 cm2. A minipump was connected to the gas inlet of the large vessel, which was placed inside the incubator, so that the incubator atmosphere was circulated through the vessel. When active gassing was employed, the productivity of the adenovirus and AAV vectors significantly increased. This vector production system was achieved by improved CO2 and air exchange and maintenance of pH in the culture medium. Viral production with active gassing is particularly promising, as it can be used with existing incubators and the large culture vessel can readily be converted for use with the active gassing system.

Anti-apoptotic and neuroprotective effects of edaravone following transient focal ischemia in rats.

To investigate the effect of an antioxidant edaravone on the apoptotic process, we examined Bax and Bcl-2 immunohistochemical expression and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) reactivity. Rat focal ischemia models were prepared by 2 h transient middle cerebral artery occlusion. Edaravone or physiological saline was intravenously administered after reperfusion. After 24 h of reperfusion, infarction volume assessments, Bax and Bcl-2 immunohistochemistry and TUNEL staining were performed as well as neurological evaluation. Cortical cerebral blood flow was not statistically different between the treatment-groups. Edaravone-treated animals showed significantly improved neurological outcome. Total and cortical infarct volumes in the edaravone group significantly decreased. In addition, edaravone-treatment provided a significant reduction in the number of TUNEL-positive apoptotic cells, a decrease in Bax immunoreactivity and an increase in Bcl-2 expression within the peri-infarct area. Edaravone shows an excellent neuroprotective effect against ischemia/reperfusion brain injury through a Bax/Bcl-2 dependent anti-apoptotic mechanism.

Differential effects of sublethal ischemia and chemical preconditioning with 3-nitropropionic acid on protein expression in gerbil hippocampus.

Pretreatment with a low dose of 3-nitropropionic acid (3-NPA) has been shown to induce ischemic tolerance in the gerbil hippocampus. It is well known that sublethal (2-min) ischemia also induces ischemic tolerance. To investigate the acquisition of ischemic tolerance with 3-NPA, we examined the protein expression after 3-NPA treatment in comparison with sublethal ischemia. Immunohistochemical studies revealed intense expression of Bcl-2 and Bcl-xL in the hippocampal CA1 area after 3-NPA treatment. Furthermore, the time course of the expression of Bcl-xL showed a similar pattern to the acquisition of ischemic tolerance by 3-NPA treatment. The induction of Bcl-xL occurred in the hippocampal CA1 area at 24 h after 3-NPA treatment, and significant induction was observed at 48 h. Western blot analysis of hippocampus harvested 48 h after the pretreatment, showed that the expression of Bcl-2 and Bcl-xL was significantly increased by either 3-NPA treatment or 2-min ischemia. However, PMCA1 and HSP70 protein expression increased only in the sublethal ischemia treated group. The difference between 3-NPA treated group and control group was not statistically significant. These results suggest that Bcl-2 and Bcl-xL are essential for acquisition of ischemic tolerance, while HSP70 and PMCA1 play important roles in the enhancement of ischemic tolerance.

Ischemic preconditioning decreases intracellular zinc accumulation induced by oxygen-glucose deprivation in gerbil hippocampal CA1 neurons.

In normal gerbils, intracellular zinc ions ([Zn2+]i) and calcium ions ([Ca2+]i) accumulate in hippocampal CA1 neurons after global ischemia. We examined whether ischemic preconditioning modifies these changes in gerbil hippocampal slices. In normal slices, large increases in [Zn2+]i and [Ca2+]i were observed in the stratum radiatum of the CA1 area after oxygen-glucose deprivation. In preconditioned slices, there were significantly decreased peak levels of [Zn2+]i and [Ca2+]i in CA1. However, there were no differences in the peak levels of these ions in CA3 and dentate gyrus. These results suggest that modified [Zn2+]i and [Ca2+]i accumulation after an ischemic insult might be important for the mechanisms of ischemic tolerance induced by preconditioning.

Heterotopic graft of infant rat brain as an ischemic model for prolonged whole-brain ischemia.

By using a heterotopic brain graft model, we have made histological and electrophysiological studies of the infant rat brain after prolonged ischemia. An infant rat head which had undergone ischemia for more than 90 min, was grafted onto an adult rat by anastomosing the thoracic vessels to the femoral vessels of the host rat. Histological and histochemical studies carried out 10 days after the operation showed that the development of the hippocampus and cerebellum in the grafted brain appeared to be normal. Interneuron growth in the hippocampus and migration of the granule cells in the cerebellum had occurred to a similar extent as in control rats. Extracellular recordings in the hippocampus showed normal characteristics of the postsynaptic potentials including long-term potentiation. This heterotopic graft model would be useful for studying brain function after long periods of ischemia.

Distinct patterns of gene transfer to gerbil hippocampus with recombinant adeno-associated virus type 2 and 5.

Genetic modification of the gerbil hippocampal neuronal cells in vivo helps us understand the mechanisms of neuronal function under various circumstances such as ischemic insult. In this study, we examined the distinct distribution of the recombinant adeno-associated virus type 2 (rAAV2) and rAAV5 vectors for gene delivery to primary cultured cells and the gerbil hippocampus. Mixed cortical cultures containing both neurons and astrocytes from E17 rat embryos were infected with rAAVs containing the Cytomegalovirus virus (CMV) promoter. rAAV2 was preferably transduced to neurons, whereas rAAV5 was inclined to be transduced to astrocytes in vitro. rAAV2 and rAAV5 vectors, each with the CMV or Rous sarcoma virus (RSV) promoter, were injected into the gerbil hippocampus using a stereotaxic apparatus. Five days after injection, transgene expression was analyzed with X-gal staining. In the gerbil hippocampus, rAAV5 with the CMV promoter achieved a higher overall transgene expression than rAAV2 with the CMV promoter. The transgene expression of rAAV2 with the RSV promoter was found in the pyramidal and granular cells, while the transgene expression of rAAV5 with the RSV promoter was preferentially found in the granular cells. These findings would be valuable in optimizing rAAV-mediated gene transfer to the gerbil hippocampus.

Upregulation of GluR2 decreases intracellular Ca2+ following ischemia in developing gerbils.

Developing animals are known to be resistant to cerebral ischemia. To investigate the mechanisms by which developing animals exhibit ischemic resistance, we examined the changes in intracellular calcium ([Ca2+]i) after oxygen-glucose deprivation (OGD) using hippocampal slices from gerbils. We found that increases of [Ca2+]i in hippocampal CA1 neurons is significantly less after OGD in developing gerbils than in adults. Western blot analysis of AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid) receptors (AMPARs) showed that GluR2 expression, but not that of the other AMPARs is significantly higher in developing gerbils than in adults. Expression of the anti-apoptotic proteins such as HSP70, Bcl-XL, and plasma membrane Ca2+-ATPase type1 (PMCA1) are not higher in the developing gerbils than in adults. These results suggest that the higher expression of GluR2 is important for the smaller increases in [Ca2+]i and enhanced resistance to ischemia-induced neuronal damage in developing animals.