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. 2018 Feb 6;24(6):508–518. doi: 10.1111/cns.12818

Expression of nerve growth factor carried by pseudotyped lentivirus improves neuron survival and cognitive functional recovery of post‐ischemia in rats

Jia‐Yu Cao 1, Yong Lin 1, Yan‐Fei Han 2, Sheng‐Hao Ding 1, Yi‐Ling Fan 1, Yao‐Hua Pan 1, Bing Zhao 1, Qin‐Hua Guo 1, Wen‐Hua Sun 1, Jie‐Qing Wan 1,✉, Xiao‐Ping Tong 2,✉
PMCID: PMC6489714  PMID: 29409115

Summary

Aims

Nerve growth factor (NGF) has been reported to prevent neuronal damage and contributes to the functional recovery in animal brain injury models and human ischemic disease as well. We aimed to investigate a potential therapeutic effect of NGF gene treatment in ischemic stroke and to estimate the functional recovery both at the cellular and cognitive levels in an ischemia rat model.

Methods

After microinjection of pseudolentivirus‐delivered β‐NGF into an established ischemic stroke model in rats (tMCAO), we estimated neuronal cell apoptosis with TUNEL labeling and neurogenesis by cell proliferation marker Ki67 staining in both ischemic core and penumbra of striatum. Furthermore, we used behavioral functional tests, Morris water maze performance, to evaluate cognitive functional recovery in vivo and propose a potential underlying mechanism.

Results

We found that pseudolentivirus‐mediated delivery of β‐NGF gene into the brain induced high expression in striatum of the infarct core area after ischemia in rats. The β‐NGF overexpression in the striatal infarction core after ischemia not only improved neuronal survival by reducing cell apoptosis and increasing cell proliferation, but also rescued cognitive functional impairment through upregulation of GAP‐43 protein expression in tMCAO rat model of ischemia.

Conclusion

This study demonstrates a potential β‐NGF gene therapy by utilization of pseudolentivirus in ischemia and indicates future applications of NGF gene treatment in ischemic patients.

Keywords: cell proliferation, cognitive functional recovery, gene therapy, ischemic stroke, nerve growth factor, neuronal apoptosis

1. INTRODUCTION

Stroke is a leading cause of mortality and morbidity worldwide. Stroke is a neural disease, clinically manifested by transient or permanent brain dysfunction. It is caused by various factors such as cerebral artery stenosis, occlusion, or rupture, and eventually induced acute cerebral blood circulation disorders.1, 2, 3 Ischemic stroke is the most common form, representing 87% of stroke cases. Due to ischemia and hypoxia, this type of stroke causes neuronal apoptosis and cell death, which ultimately leads to the loss of brain functions. Currently, intra‐arterial (IA) device‐based therapy in multiple clinical trials has demonstrated efficacy for the treatment of acute ischemic stroke in patients. However, the short time window for surgery within 6 hours of stroke onset limits the successful rate in patients’ treatment.4, 5 For the majority of stroke patients, especially combined with complex syndromes, searching for an alternative drug administration or gene therapy becomes a serious and urgent issue.

Nerve growth factor (NGF) is the first identified and best characterized member of the neurotrophin family. Nerve growth factor (NGF) is an important regulator of neural survival, development, plasticity, known to play an important role in stimulating and modulating the differentiation as well as the maturation of developing and adult neurons in the central and peripheral nervous systems.6, 7, 8, 9, 10 Exogenous administration of NGF has also been reported to prevent neuronal damage and contributes to the functional recovery after brain injury in animal brain injury models and human ischemia disease.11, 12, 13, 14 However, as NGF is relatively impermeable to the blood‐brain barrier (BBB), it becomes difficult to accurately estimate the efficiency of reaching to the damaged brain lesion area by intramuscular injection.15 The high dosages of NGF administration which are usually at microgram amounts are known to induce remarkable side effects such as aberrant sympathetic neurite sprouting, reduced weight gain and food intake.16, 17, 18 Moreover, the invasive strategies of the NGF administration, for instance, intraventricular infusion,14 could induce tissue damage, hemorrhage, hyperalgesia, and infections in clinical trials.19, 20 In our previous study, we first cloned and identified the gene of β‐NGF which encodes one of three NGF subunits and can exhibit all biological activities of the NGF.21 Through microinjection of β‐NGF carried by pseudolentivirus into the injured hippocampus after traumatic brain injury (TBI), β‐NGF can last as long as 3 weeks in the brain and further rescued the impaired cognitive function of rats after TBI.21 It suggests a potential application for pseudolentivirus‐delivered NGF gene therapy in neuronal diseases.

To directly investigate a potential therapeutic NGF gene treatment in ischemic stroke disease, we used an established ischemic stroke model tMCAO in rats22, 23 and estimated the effects on neuronal functional recovery in infarction core by pseudolentivirus‐delivered β‐NGF gene into the ischemic brain. We found that microinjection of β‐NGF in striatum not only reduced the number of cell apoptosis by TUNEL labeling in both ischemic core and penumbra, but neurogenesis was also greatly increased in medial‐ventral striatum characterized by cell proliferating marker Ki67. Furthermore, the results of the Morris water maze behavior test supported that β‐NGF overexpression in ischemic rat brain rescued the cognitive impairment which was largely mediated by upregulation of growth‐associated protein 43 (GAP‐43). Growth‐associated protein 43 (GAP‐43) is a nervous tissue‐specific cytoplasmic protein and plays a key role in neurite formation and synaptic plasticity.24, 25 Hence, multiple brain functional evaluations illustrate de novo NGF gene therapy in ischemic disease.

2. MATERIALS AND METHODS

2.1. Animals

All animal procedures complied with the animal care standards set forth by the US National Institutes of Health and were approved by the Institutional Animal Care and Use Committee, Shanghai Jiao Tong University School of Medicine. Male Sprague‐Dawley rats (280‐300 g) were purchased from Shanghai Jie‐Si‐Jie Animal Co., Ltd. (Shanghai, China) and housed under a 12‐12‐hour light‐dark cycle with food and water provided ad libitum from the cage lid.

2.2. Middle cerebral artery occlusion (MCAO) model in rat

Rat brain ischemic model was established as described before and slightly modified.26, 27 In brief, six‐ to 8‐week‐old male SD rats were anaesthetized with an intraperitoneal injection of chloral hydrate (350 mg/kg). A suture of 0.26 millimeters (mm) in diameter with a 0.36‐mm‐diameter tip (Sha‐Dong Biological Technology Co., Ltd., Beijing, China) was inserted into the internal carotid artery (ICA) through a cut of the common carotid artery (CCA) to occlude the middle cerebral artery (MCA). Reperfusion was allowed after 45 minutes by monofilament removal. Regional cerebral blood flow was monitored by laser Doppler flowmetry (VMS‐LDF2, Moor Instruments Ltd., UK), and rats with <20% reduction in cerebral blood flow at the core regions of the MCA territory were excluded from the study. After reperfusion for 24 hours, animals were sacrificed, and infarct volume in the brain was determined by TTC staining.

2.3. Construction of pseudo lentivirus‐carrying β‐NGF fusion gene

The pseudo lentivirus‐carrying β‐NGF gene, fused with green fluorescent protein gene (Lv‐NGF‐GFP), was constructed as mentioned in our previous work.21 In brief, the β‐NGF gene was cloned into the modified lentiviral vector of the pLVX‐IRES‐ZsGreen1 (Clontech, Mountain View, CA, USA), which was cotransfected into the 293T cells with the packaging plasmid mixes (Clontech, Mountain View, CA, USA). The pseudo lentiviruswith GFP in the absence of β‐NGF (Lv‐GFP) was used as sham control.

2.4. Lv‐NGF‐GFP/Lv‐GFP microinjection in ischemic rat brain

Experimental ischemic rats were divided into 2 groups: Lv‐NGF‐GFP transfected tMCAO group and Lv‐GFP transfected tMCAO group. In short, 5 μL (~1 × 108 TU/mL) of the lentivirus‐carrying NGF‐GFP or GFP was injected into the striatum of the ipsilateral hemisphere of the rat brain after the rat underwent ischemic surgery. For striatum injection site, coordinates were 1.8 mm anterior to bregma, 3 mm from the midline, and 5 mm deep from the dura. After 2‐3 weeks of virus transfection, the animals were sacrificed and utilized for immunohistochemistry and Western blotting experiments.

2.5. Immunohistochemistry and Western blotting

Twenty‐four hours after lentivirus‐transfected rats underwent ischemic surgery, they were perfused with 4% paraformaldehyde (PFA) in PBS for 15 minutes. Then the brain was removed and postfixed in 4% PFA at 4°C for 4 hours, followed by cryoprotection in concentrations of 20% and 30% sucrose in PBS at 4°C for another 3 days. Twenty micrometers (μm) thickness sections of the brain was cut using a cryostat (Leica Microsystems, Wetzlar, Germany). Sections were then prepared for immunohistochemistry. The following primary antibodies used for immunohistochemistry include the following: anti‐NeuN antibody (Millipore, CBL242, 1:500), anti‐Ki67 antibody (Abcam, ab15580, 1:500), TUNEL Kit (REF 12156792910, Germany), and anti‐S100β (Sigma‐Aldrich, AMAB91038, 1:500). The secondary antibodies included the following: Goat anti‐Rabbit Alexa Fluor 647 (Thermo Fisher Scientific, A27040), Goat anti‐Mouse Alexa Fluor 647 (Thermo Fisher Scientific, A32728). Sections were incubated with DAPI for 30 minutes at room temperature and mounted on glass slides in Fluoromount™ Aqueous Mounting Medium (Sigma, F4680). All slice images were acquired on a Leica TCS SP8 confocal microscope with HC PL APO CS2 20x/0.75 DRY objective. Image analysis was performed by Image‐Pro Plus (Media Cybernetics). The images of different channels were thresholded, and cell numbers were determined according the DAPI channel threshold image.

For Western blotting, the ischemic hemisphere of rat brain was cut in half: anterior part and posterior part. The anterior part, in which the injection site was located, was only used for β‐NGF gene expression. In brief, the ipsilateral part of the rat brain was homogenized in the lysis buffer (50 mmol/L Tris HCl, pH 8.0, 150 mmol/L NaCl, 1% NP‐40, 0.5% deoxycholate, and 0.1% SDS). After the tissue lysate was centrifuged, the supernatant was stored. The BCA protein assay kit was used to measure the protein concentration of the supernatant. The proteins were analyzed for the expression of the β‐NGF gene. Hundred micrograms (μg) of proteins for each sample was loaded and separated on a 15% Tris‐glycine gel. The proteins were transferred into a PVDF membrane, which was then blocked in 5% nonfat dry milk in PBS with 0.05% Tween‐20 (PBS‐T) at 4°C, overnight. After which, the membrane was incubated with goat anti‐β‐NGF primary antibody (Ab) (1:250, Sigma‐Aldrich, St. Louis, MO, USA) or with rabbit anti‐GAP43 Ab (1:10 000, Abcam, Cambridge, MA, USA), at room temperature (RT) for 1 hour. The blot was washed with PBS‐T for 10 minutes, 3 times and then incubated in Peroxidase AffiniPure Donkey anti‐Goat IgG (1:5000, Jackson ImmunoResearch, West Grove, PA, USA) or in Peroxidase‐conjugated AffiniPure Goat Anti‐Rabbit IgG (H + L) at RT for 1 hour. After PBS‐T washing, the blot was incubated with Immobilon Western Chemiluminescent HRP substrate (EMD Millipore, Billerica, MA, USA). β‐actin was used as an internal control of each sample by incubation with HRP‐conjugated anti‐β‐actin IgG (1:5000, Proteintech Group, Chicago, USA). Densitometric analysis was performed using the NIH Image Program (ImageJ 1.43u) to calculate the ratio of the β‐NGF density to β‐actin density.

2.6. Morris water maze assessment

The evaluation of the cognitive function in the rat was performed with the Morris water maze from the 14 days until the 20 days, after the lentivirus injection and brain ischemia as described in our previous study.21 Briefly, the rats were tested in a black water maze. A black platform with a diameter of 12 and 2 cm lower than the water level was placed in the maze pool. The maze pool was divided into four equal quadrants. The rats were tested for swimming in each quadrant and rested for 4 minutes before the next round test was started. A 60‐second time limit was set up for each rat to swim to the platform. If the rat failed to find it, it was placed on the platform to rest for 10 seconds. The escape latency and the length of swimming path of the rat were collected for analysis.

2.7. Statistical analysis

The data in this study were analyzed using one‐way ANOVA (for three or more samples) followed by post hoc Tukey‐Kramer multiple comparisons test. Statistical analysis was carried out using GraphPad InStat (GraphPad Software). For all biochemistry and immunohistochemistry experiments, n values represent the number of rats. Results are presented as mean ± SEM, and statistical significance was set at *P < 0.05, **P < 0.01.

3. RESULTS

3.1. Establishment of tMCAO model of rat ischemia

First, we established a transient middle cerebral artery occlusion (tMCAO) ischemic model in rats. As shown in the cartoon and diagram of MCAO procedures (Figure 1A,B), the rat artery was occluded for 45 minutes, and the brain was reperfused for 24 hours. A series of brain sections were obtained and exhibited 2,3,5‐triphenyltetrazolium chloride (TTC) staining to detect the extent of the tissue damage as shown in Figure 1C. From the TTC staining images in ipsilateral occlusion side, we could observe the hypoxia‐induced ischemic injury that occurred in both cortex and striatum (Figure 1C), which were identified as the two major infarction core regions in the brain.

Figure 1.

Figure 1

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Establishment of transient middle cerebral artery occlusion (tMCAO) model of rat ischemia. A‐B, The cartoon (A) and experimental procedure diagram (B) show a tMCAO model. In brief, a 6‐ to 8‐wk‐old male SD rat was anaesthetized, and a suture of 0.26 mm in diameter with a 0.36‐mm‐diameter tip was inserted into the internal carotid artery (ICA) through a cut of the common carotid artery (CCA) to occlude the middle cerebral artery (MCA). Reperfusion was allowed after 45 min by monofilament removal. C, triphenyltetrazolium chloride (TTC) staining images in ipsilateral occlusion side, the hypoxia‐induced ischemic injury mostly occurred in both cerebral cortex and striatum in serial sections of tMCAO rat brain

3.2. Pseudo lentivirus‐carrying β‐NGF microinjection in striatum of ischemic rat brain

In our previously published study, we first cloned and identified the gene β‐NGF, which encodes one of three NGF subunits and can exhibit all biological activities of NGF.21 To investigate whether NGF has an impact on ischemia, the pseudo lentivirus‐carrying β‐NGF gene, fused with green fluorescent protein gene (Lv‐NGF‐GFP), was stereotaxically injected into the dorsal‐lateral striatum (Figure 2A,B). Lv‐NGF‐GFP was successfully fused into striatal neurons after 2 weeks as GFP fluorescence‐labeled cells were mostly colocalized with the neuronal nuclear marker NeuN but not with glial cell's markers such as astrocytic marker S100β (Figure 2C,D). Western blotting results further validated a significant increase of NGF protein expression in pre‐injected Lv‐NGF‐GFP group compared with that in both Lv‐GFP injection group and untreated tMCAO sham group, respectively. For Lv‐NGF‐GFP injection, the relative NGF protein expression level was increased about 2‐fold compared with that in LV‐GFP transfection alone and tMCAO sham group (Lv‐NGF‐GFP: 2.025 ± 0.26, n = 3 rats; Lv‐GFP: 0.996 ± 0.16, n = 3 rats; tMCAO sham: 1.00 ± 0.15, n = 3 rats) (Figure 2E). The statistical P values for ANOVA followed by Tukey‐Kramer multiple comparisons test were as follows: P < 0.05, Lv‐NGF‐GFP vs Lv‐GFP; P < 0.05, Lv‐NGF‐GFP vs tMCAO sham group; P > 0.05, Lv‐GFP vs tMCAO sham group.

Figure 2.

Figure 2

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Pseudolentivirus‐mediated β‐NGF delivery into the striatum of ischemic rat brain. A, The cartoon illustrates the lentiviral constructs used and the general protocol for viral delivery into the striatum of adult rats. B, Representative images show the location of β‐NGF delivery identified with GFP fluorescence in the dorsal striatum. Scale bar, 50 μm. C, Representative images show the colocalization of Lv‐NGF‐GFP with neuronal nuclear marker NeuN, which indicates most striatal neurons were successfully transfected with β‐NGF. The arrows show the colocalization of neuronal marker NeuN with GFP fluorescence. Scale bar, 20 μm. D, Same as in (C) but for astrocytic marker S100β labeling. It clearly shows no colocalization of astrocytic marker S100β with GFP fluorescence. Scale bar, 20 μm. E, Representative Western blot (left panel) and quantitative analysis (right panel) for NGF expression in transient middle cerebral artery occlusion (tMCAO) sham group, Lv‐GFP tMCAO group and Lv‐NGF‐GFP tMCAO group, respectively. Quantified results are normalized to β‐actin expression. A significant increase of NGF protein expression in Lv‐NGF‐GFP tMCAO group is shown in the representative image. Values are normalized to Sham group and represent mean ± SEM n = 3 rats per group. *P < 0.05 Lv‐NGF‐GFP vs Sham or Lv‐GFP group using ANOVA Turkey‐Kramer multiple comparisons test

3.3. β‐NGF transfection reduces cell apoptosis and increases neurogenesis in infarct striatum after tMCAO in rats

It is well known that striatum is one of the infarction core brain regions in tMCAO and experiences cell damage and cell loss at the early onset of ischemia. Indeed, when we used TUNEL to label cell apoptosis which signals cell death progression, we found TUNEL‐labeled cells were largely restricted to the ischemic core and penumbra of dorsal striatum, and most TUNEL‐labeled cells were colocalized with the neuronal nuclear marker NeuN (Figure 3A,B). Due to the striatum being severely damaged after postischemia, it was hard to distinguish single TUNEL‐labeled–positive cells in the striatal infarction core area. Thus, we calculated the apoptotic cell numbers by choosing the penumbra of the infarct region. As shown in the areas between the white‐dotted lines among the three groups in Figure 3A‐C, the TUNEL‐labeled cells in Lv‐NGF‐GFP group were dramatically reduced compared with those in Lv‐GFP group and sham group, respectively. The total TUNEL‐positive cell numbers in the penumbra regions were as follows: Lv‐NGF‐GFP: 11 ± 0.58/mm2, n = 3 rats; Lv‐GFP: 26.67 ± 1.33/mm2, n = 3 rats; tMCAO sham: 25.67 ± 1.76/mm2, n = 3 rats (Figure 3D). The statistical P values for ANOVA followed by Tukey‐Kramer multiple comparisons test were as follows: P < 0.01, Lv‐NGF‐GFP vs Lv‐GFP; P < 0.01, Lv‐NGF‐GFP vs tMCAO sham group; P > 0.05, Lv‐GFP vs tMCAO sham group. Therefore, this observation indicated that NGF overexpression contributed to the improvement of cell survival in ischemia.

Figure 3.

Figure 3

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β‐NGF overexpression reduces cell apoptosis in infarct striatum after transient middle cerebral artery occlusion (tMCAO) in rat. A‐C, Representative images show immunohistochemistry for cell apoptosis marker TUNEL in infarct striatum after postischemia. A significant decrease of neuronal cell death in the region of ischemic penumbra of striatum as shown in the region between the two white‐dotted lines after Lv‐NGF‐GFP overexpression. The images are collected from 3 rats in each group. Scale bars, 20 μm. D, The graph summarizes total TUNEL‐labeled cells per mm2 in the striatal ischemic penumbra of tMCAO sham group, Lv‐GFP tMCAO group and Lv‐NGF‐GFP group. Values are represented as mean ± SEM n = 3 rats per group. **P < 0.01 Lv‐NGF‐GFP vs Sham or Lv‐GFP group using ANOVA Turkey‐Kramer multiple comparisons test

Nerve growth factor (NGF) microinjections in the brain reduced cell apoptosis in tMCAO, and interestingly, we further found a dramatic increase of neurogenesis in the medial‐ventral striatum which was slightly away from the ischemic infarct core and penumbra regions (Figure 4A‐C). Labeling with the cell proliferation marker Ki67, we counted the Ki67‐positive cell numbers in Lv‐NGF‐GFP, Lv‐GFP, and tMCAO sham group. The total proliferating cell numbers in these three groups were as follows: Lv‐NGF‐GFP: 35.33 ± 2.85/mm2, n = 3 rats; Lv‐GFP: 18.33 ± 1.67/mm2, n = 3 rats; tMCAO sham: 17.00 ± 0.58/mm2, n = 3 rats (Figure 4D). The statistical P values for ANOVA followed by Tukey‐Kramer multiple comparisons test were as follows: P < 0.01, Lv‐NGF‐GFP vs Lv‐GFP; P < 0.01, Lv‐NGF‐GFP vs tMCAO sham group; P > 0.05, Lv‐GFP vs tMCAO sham group.

Figure 4.

Figure 4

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β‐NGF overexpression increases neurogenesis in infarct striatum after transient middle cerebral artery occlusion (tMCAO) in rat. A‐C, Representative images show immunohistochemistry for cell proliferating marker Ki67 in infarct striatum after postischemia and a significant increase of neurogenesis in Lv‐NGF‐GFP transfected group compared with that in Lv‐GFP transfected tMCAO group or in tMCAO sham group. The images were collected from 3 rats for each group, scale bars, 20 μm. D, The statistical graph shows total Ki67‐labeled cells per mm2 in striatum of tMCAO sham group, Lv‐GFP tMCAO group and Lv‐NGF‐GFP group. Values are represented as mean ± SEM n = 3 rats per group. **P < 0.01 Lv‐NGF‐GFP vs Sham or Lv‐GFP group by ANOVA Turkey‐Kramer multiple comparisons test

3.4. β‐NGF upregulation of GAP‐43 promotes cognitive functional recovery

It is known that NGF plays a critical role in maintaining functional connections in adult brain neurons.8, 9, 10, 11, 12, 13, 21 In a developing brain, low synthesis of NGF can negatively affect neurogenesis and neuroplasticity.7, 28 To further assess the mechanism of neuroprotection by NGF after postischemia in rat, we investigated a potential intracellular signaling pathway, that is, growth‐associated protein 43 (GAP‐43), which mediates neuronal apoptosis and neurogenesis.24, 25, 29, 30 As shown in Figure 5A, we found that the basal GAP‐43 protein levels were very low in both tMCAO sham group and Lv‐GFP transfected tMCAO group. However, a 2.1‐fold and 2.5‐fold increase of GAP‐43 protein levels occurred in Lv‐NGF‐GFP transfected tMCAO group when compared with that in tMCAO sham control and Lv‐GFP transfection group. The statistical P values were < 0.01 for Lv‐NGF‐GFP group compared with either Lv‐GFP or tMCAO sham group.

Figure 5.

Figure 5

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β‐NGF upregulation of GAP‐43 promotes cognitive functional recovery in transient middle cerebral artery occlusion (tMCAO) rats. A, Representative Western blots (left panel) and quantitative analysis (right panel) for GAP‐43 protein expression in tMCAO sham group, Lv‐GFP tMCAO group and Lv‐NGF‐GFP tMCAO group, respectively. Quantitative GAP‐43 protein expression is normalized to β‐actin. A significant increase of GAP‐43 in Lv‐NGF‐GFP tMCAO group is shown in the representative image. Values are normalized to sham group and represent mean ± SEM. n = 3 rats per group. **P < 0.01 Lv‐NGF‐GFP vs Sham or Lv‐GFP group using ANOVA Turkey‐Kramer multiple comparisons test. B, The escape latencies of Morris water maze performance are analyzed during the examined days after lentivirus transfection in both tMCAO and wild‐type control rats. As shown in the graph, Lv‐GFP transfected tMCAO group shows apparent impaired working learning memory compared with wild‐type control group (P < 0.05, ANOVA post hoc test, n equals 6 for Lv‐GFP group and 8 for wild‐type control group). In contrast, Lv‐NGF‐GFP group shows improved functional recovery compared with that of Lv‐GFP group starting at Day 16 after lentivirus transfections in tMCAO (*P < 0.05, Lv‐NGF‐GFP group vs Lv‐GFP group at Day 16, 17, 19, 20). Lv‐NGF‐GFP group shows no significant difference from wild‐type control group after daily examination from day 16 (## P < 0.01, Lv‐NGF‐GFP group vs wild‐type control at Day 15). C, The lengths of swimming path of Morris water maze performance were analyzed during the examined days after lentivirus transfection in tMCAO rats and wild‐type control rats. As shown in the graph, Lv‐GFP tMCAO group shows significantly impaired working learning memory compared with that in wild‐type control (P < 0.05, ANOVA post hoc test, n equals 6 for Lv‐GFP tMCAO group and 8 for wild‐type control group). In contrast, Lv‐NGF‐GFP tMCAO group shows significant functional improvement compared with that in Lv‐GFP tMCAO group starting at Day 15 after lentivirus transfection (*P < 0.05, Lv‐NGF‐GFP group vs Lv‐GFP group at Day 15, 19, 20; **P < 0.01, Lv‐NGF‐GFP group vs Lv‐GFP group at Day 16, 17, 18. ANOVA post hoc test, n equals 5 for Lv‐NGF‐GFP group and 6 for Lv‐GFP group)

During neuronal development or regeneration from brain injury, the expression of GAP‐43 can fluctuate from very low levels in resting neurons to high levels in cells undergoing axonogenesis or synaptic plasticity.24, 31 Therefore, the significantly elevated expression of GAP‐43 mediated by NGF in our study prompted us to further estimate the cognitive functional recovery in postischemic rats. Morris water maze (MWM) task has often been used in the validation of neurocognitive disorders and the evaluation of possible neurocognitive treatments in rodent models.32 The lesions in distinct brain regions such as hippocampus, striatum, cerebral cortex, and cerebellum are usually linked to impaired MWM performance. Therefore, we next investigated whether NGF overexpression could improve or rescue impaired cognitive functions after tMCAO in rats. We measured the standard performance during the acquisition phase of MWM task, including of escape latency and the total length of the swimming path. To our surprise, the escape latencies in Lv‐NGF‐GFP transfected tMCAO rats recovered to almost the normal range after 16 days for NGF microinjections when compared with wild‐type rats (Figure 5B). On the contrast, Lv‐GFP transfected tMCAO rats still showed distinct impairments in escape latency compared with wild‐type rats (P < 0.05 for each day point). The Lv‐NGF‐GFP group also showed significant improvement of cognitive functions after day 16 compared with Lv‐GFP group except at the examined day 18 (Figure 5B). A similar phenomenon was observed in the length of the swimming path of MWM task. As shown in Figure 5C, there was no significant difference in the total swimming distances between Lv‐NGF‐GFP tMCAO group and wild‐type control group at each time point, which indicated NGF overexpression greatly improved an impaired working memory in ischemic rats. Moreover, Lv‐GFP transfected tMCAO group did not show any improvement when compared with NGF‐transfected tMCAO group since day 15 (Figure 5C). Taken together, this data suggested that NGF‐induced upregulation of GAP‐43 promoted neuronal axonogenesis and synaptic remodeling and eventually contributed to the cognitive functional recovery in ischemic rat model.

4. DISCUSSION

Since the gene delivery method into the brain was first reported in 1998, lentiviral‐delivered gene therapy has been applied for the treatment of several neural disorders, such as Alzheimer Disease, Parkinson's disease, and traumatic brain injury (TBI).21, 33, 34, 35, 36 In our study, we evaluated the functional role of NGF carried by pseudolentivirus in the treatment of ischemic disease. By utilizing a well‐established ischemic stroke model, tMCAO in rats, we found that lentiviral delivery of β‐NGF into the striatum not only reduced the number of apoptotic cells as shown by TUNEL staining in both ischemic core and penumbra, but neurogenesis was also greatly increased in medial‐ventral striatum identified by cell proliferating marker Ki67. Furthermore, we demonstrated that overexpression of NGF rescued ischemic rats’ cognitive impairment through upregulation of GAP‐43. Hence, multiple brain functional assays identified a novel NGF gene therapy for ischemic disease.

One of the mechanisms of neuron repair and functional recovery induced by NGF is largely through the regulation of stability of GAP‐43.21, 24, 25, 29, 30, 31 GAP‐43, also known as F1, B‐50 or neuromodulin, was identified as a synaptic phosphoprotein regulated by Ca2+ and various peptides in the mid‐1970s.37, 38 GAP‐43 in the adult brain retains a capacity of synaptic reorganization in response to abnormal patterns of physiological activity. During the course of neuronal development or regeneration, the expression of GAP‐43 can reach to over 100‐fold range.24, 31 High levels of GAP‐43 protein expression in injured brain related to repair processes lend support to a morphological basis for long‐term memory and synaptic plasticity.24 Our current results showed that lentiviral delivery of NGF induced as high as 2‐fold increase of GAP‐43 proteins in ischemic infarction core, which indicated that an initial brain functional recovery occurred. Indeed, application of cognitive functional tests such as the Morris water maze further proved a rescue of learning and memory impairment in NGF‐transfected tMCAO rats.

As a neurotrophin family member, NGF expresses broadly in both central nervous system (CNS) and peripheral nervous system (PNS).6, 7 Previously published studies have shown that NGF plays an important role for neuronal survival, maturation, functional repair, and synaptic plasticity.6, 7, 8, 9, 10, 21 In our present study, we provided more evidence that NGF promoted cell survival by reducing cell apoptosis and increasing cell proliferation in tMCAO rat brain. Given the complex downstream signaling pathways that NGF could act upon for neuronal repair in the brain,7, 21, 24, 25, 28, 29, 30, 31 the possible mechanisms by which overexpressed NGF rescues neuronal damage and cognitive function includes two major intrinsic signals and could be favored in our experimental ischemic model. One is to regulate specific receptor tropomyosin kinase receptor A (TrkA) and p75 pan‐neurotrophin receptor (p75NTR) complex.39, 40, 41 Through TrkA/p75NTR complex activation, ras‐mitogen–activated protein kinase (MAPK), extracellular signal‐regulated kinase (ERK), phosphatidylinositol 3‐kinase (PI3K)‐Akt, and phospholipase C (PLC) downstream were potentially activated.42, 43, 44 Second, NGF could directly bind to p75NTR, which activates nuclear factor‐κB (NFκB) pathway, independent of TrkA.45 These signaling pathways can promote cell survival, cell proliferation, and/or cell differentiation. Taken together, there is a great possibility that lentiviral‐delivered exogenous NGF exerts vital effects on cell survival and neurogenesis in situ after tMCAO in rats.

Last but not least, increased understanding and development of new methodologies for gene delivery and gene editing have become one of the most promising approaches to treat genetic diseases, viral diseases, cancers, and brain disorders.46, 47, 48, 49 The utilization of gene therapies over traditional drug administration through intramuscular injection or intraventricular infusion is clearly beneficial. For instance, adenosine‐associated virus (AAV) delivery gene method, zinc‐finger nucleases (ZFN), and clustered regularly interspaced short palindromic repeats (CRISPR) gene editing techniques could unveil more advantages for accurate targeting of altered/mutant genome with rapid repair characteristics and less side effects in the future treatment of human diseases.

CONFLICT OF INTEREST

The authors declare no conflict of interest.

ACKNOWLEDGMENTS

This work was supported by Grants from the Ren Ji Hospital Cultivation Foundation (No. RJZZ15‐004), National Science and Nature Committee of China (No. 81471244), Natural Science Foundation of Shanghai Jiao Tong University School of Medicine (No. 14XJ10021), National Natural Science Foundation of China (No. 31571063 and No. 91632104), the program for Professor of Special Appointment (Eastern Scholar for Dr. X.T.) at Shanghai Institutions for Higher Learning (1510000084) and Shanghai Pujiang Program (15PJ1404600).

Cao J‐Y, Lin Y, Han Y‐F, et al. Expression of nerve growth factor carried by pseudotyped lentivirus improves neuron survival and cognitive functional recovery of post‐ischemia in rats. CNS Neurosci Ther. 2018;24:508–518. 10.1111/cns.12818

Jia‐Yu Cao and Yong Lin are contributed equally to this work.

Contributor Information

Jie‐Qing Wan, Email: Jieqingwan@126.com.

Xiao‐Ping Tong, Email: Xtong@shsmu.edu.cn.

REFERENCES

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