[Effect of injecting mouse nerve growth factor in different ways on motor development, cerebral hemodynamics and biochemical metabolism in children with cerebral palsy].

OBJECTIVE: To compare the effect of acupoint injection and intramuscular injection with mouse nerve growth factor (mNGF) on gross motor function development of children with cerebral palsy (CP), and explore the treatment mechanism. METHODS: A total of 63 children with CP were randomly divided into an observation group (32 cases, 4 cases dropped off ) and a control group (31 cases, 3 cases dropped off). Based on the routine rehabilitation therapy, the control group received intramuscular injection of mNGF(18 µg/2 mL), and the observation group received acupoint injection of mNGF at Xinshu (BL 15), Ganshu (BL 18), Pishu (BL 20), Shenshu (BL 23), Sanjiaoshu (BL 22), Shenting (GV 24), Baihui (GV 20), Fengfu (GV 16), Dazhui (GV 14), etc. Of them, 5-6 acupoints alternately were selected each time, and each acupoint was given 0.3-0.5 mL, totally 18 µg/2 mL. Both treatment were carried out once every other day for six months. Before and after treatment, the children's development of brain function was assessed using gross motor function classification system (GMFCS). Before treatment (T0), after 2 (T2), 4 (T4) and 6 (T6) months of treatment, the motor function was evaluated by gross motor function measure (GMFM-88). The systolic peak velocity (Vs), mean velocity (Vm) and vascular resistance index (RI) of anterior cerebral artery (ACA) and middle cerebral artery (MCA) were measured, and the level of N-acetyl aspartate acid (NAA), choline (Cho), lactate (Lac) and creatine (Cr) from the basal ganglia, thalamus and periventricular white mater were detected by magnetic resonance spectroscopy (MRS) technology with MAGNETOM Skyra3.0T magnetic resonance imaging system before and after treatment. RESULTS: Compared with before treatment, the GMFCS classification of the observation group after treatment was significantly improved (P<0.05); after treatment, the difference of GMFCS classification between the two groups was not significant (P>0.05), however, the observation group had a 3.142 times of feasibility for good gross motor function development by more than level 1 compared to the control group (P<0.05). After 2, 4, and 6 months of treatment, the GMFM-88 scores of the two groups showed an upward trend (P<0.01), and the increase of the observation group was greater than that of the control group (P<0.05). Compared with before treatment, in the ACA and MCA, the Vs and Vm increased, RI decreased in both groups after treatment (P<0.01), and in the brain, NAA/Cr increased, Cho/Cr and Lac/Cr decreased (P<0.01), and after treatment, the Vs, Vm of ACA and MCA and NAA/Cr of brain in the observation group were higher than those in the control group (P<0.05), and the RI of ACA and MCA and Cho/Cr and Lac/Cr of brain in the observation group were lower than those in the control group (P<0.05). CONCLUSION: The mNGF acupoint injection has a better effect on the gross motor function in the children with cerebral palsy compared with the intramuscular injection, and the mechanism may be associated with exhibiting the double effects of acupoint effect and the targeting therapy of drug, which can effectively improve the cerebral hemodynamics and the metabolism of cerebral nervous substances.

Nasal delivery of nerve growth factor rescue hypogonadism by up-regulating GnRH and testosterone in aging male mice.

BACKGROUND: Nerve growth factor (NGF) plays essential roles in regulating the development and maintenance of central sympathetic and sensory neurons. However, the effects of NGF on hypogonadism remain unexplored. METHODS: To assess the effects of NGF on hypogonadism, we established a convenient and noninvasive way to deliver NGF to the hypothalamus by spraying liposome-encapsulated NGF into the nasal cavity. The ten-month-old aging male senescence accelerate mouse P8 (SAMP8) mice with age-related hypogonadotrophic hypogonadism were used to study the role of NGF in hypogonadism. The age-matched accelerated senescence-resistant mouse R1 (SAMR1) served as a control. The ten-month-old SAMP8 mice were treated with NGF twice per week for 12 weeks. Sexual hormones, sexual behaviors, and fertility were analyzed after NGF treatment. And the mechanisms of NGF in sex hormones sexual function were also studied. FINDINGS: NGF could enhance the sexual function, improve the quality of the sperm, and restore the fertility of aging male SAMP8 mice with age-related hypogonadism by activating gonadotropin-releasing hormone (GnRH) neuron and regulating secretion of GnRH. And NGF regulated the GnRH release through the PKC/p-ERK1/2/p-CREB signal pathway. INTERPRETATION: These results suggest that NGF treatment could alleviate various age-related hypogonadism symptoms in male SAMP8 and may be usefulness for age-related hypogonadotrophic hypogonadism and its related subfertility. FUND: National Natural Science Foundation of China, Natural Science Foundation of Guangdong Province, the Science and Technology Plan Project of Guangzhou, Wenzhou Science & Technology Bureau, Guangdong Province Pearl River Scholar Fund, Guangdong province science and technology innovation leading Scholar Fund.

Organotypic Spinal Cord Culture: a Proper Platform for the Functional Screening.

Recent improvements in organotypic slice culturing and its accompanying technological innovations have made this biological preparation increasingly useful ex vivo experimental model. Among organotypic slice cultures obtained from various central nervous regions, spinal cord slice culture is an absorbing model that represents several unique advantages over other current in vitro and in vivo models. The culture of developing spinal cord slices, as allows real-time observation of embryonic cells behaviors, is an instrumental platform for developmental investigation. Importantly, due to the ability of ex vivo models to recapitulate different aspects of corresponding in vivo conditions, these models have been subject of various manipulations to derive disease-relevant slice models. Moreover spinal cord slice cultures represent a potential platform for screening of different pharmacological agents and evaluation of cell transplantation and neuroregenerative materials. In this review, we will focus on studies carried out using the ex vivo model of spinal cord slice cultures and main advantages linked to practicality of these slices in both normal and neuropathological diseases and summarize them in different categories based on application.

[Neonatal hypoxic ischemic encephalopathy treated with acupuncture combined with acupoint injection: a randomized controlled trial].

OBJECTIVE: To observe the clinical efficacy difference in the treatment of neonatal hypoxic ischemic encephalopathy (NHIE) between the combined therapy of acupuncture land acupoint injection of nerve growth fac-tor (NGF) and the combined therapy of the intravenous drop of citicoline sodium and intramuscular injection of NGF. METHODS: Sixty-one cases of NHIE were randomized into an observation group (32 cases) and a control group (29 cases). In the observation group, acupuncture combined with acupint injection of NGF was adopted. The acupoints were Baihui (GV 20), Dazhui (GV 14) and Fengfu (GV 16), 0. 5 mL each acupoint, once a day. In the control group, the intravenous drop of citicoline sodium and intramuscular injection of NGF were used. The, treatment of 7 days made one session and 3 sessions were required. Separately, in 7, 14 and 21 days of treatment, the clinical efficacy, behavior nerve determination score and adverse reaction were observed in the two groups. RESULTS: In 7, 14 and 21 days of treatment, the total effective rates were 50. 0% (16/32), 68. 8% (22/32) and 78. 1% (25/32) in the observation group and were 69. 0% (20/29), 72. 4% (21/29) and 82. 8% (24/29) in the control group. The result in the control group was better than that in the observation group in the 7 days (P< 0. 05). The behavior determination scores in 7, 14 and 21 days were all improved obviously as compared with those before treatment in the two groups (P<0. 05, P<0. 01). The score in the control group was better than that in the, observation group in the 7 days (P<0. 05). The differences were not significant in the scores between the two groups in 14 and 21 days (both P>0. 05). The incidence of adverse reaction in the observation group was lower than that in, the control group [14. 3% (5/35) vs 31. 4% (11/35), P<0. 05] and the severity was mild. CONCLUSION: In the treatment of NHIE with the combined therapy of acupuncture and acupint injection, the overall efficacy in 1 week; is inferior to that of the combined therapy of the intravenous drop of citicoline sodium and intramuscular injection of NGF. But the efficacy is improved gradually along with the treatment progression and the adverse reaction is less.

Dual growth factor-immobilized microspheres for tissue reinnervation: in vitro and preliminary in vivo studies.

Growth factors (GFs) (basic fibroblast growth factor (bFGF) and/or nerve growth factor (NGF))-immobilized polycaprolactone (PCL)/Pluronic F127 microspheres were prepared using an isolated particulate-melting method and the sequential binding of heparin and GFs onto the microspheres. The GFs immobilized on the microspheres were released in a sustained manner over 28 days, regardless of GF type. From the in vitro culture of muscle-derived stem cells, it was observed that the NGF-immobilized microspheres induced more neurogenic differentiation than the bFGF-immobilized microspheres, as evidenced by a quantitative real-time polymerase chain reaction using specific neurogenic markers (Nestin, GFAP, ß-tubulin, and MAP2) and Western blot (Nestin and ß-tubulin) analyses. The dual bFGF/NGF-immobilized microspheres showed better neurogenic differentiation than the microspheres immobilized with single bFGF or NGF. From the preliminary animal study, the dual bFGF/NGF-immobilized microsphere group also showed effective nerve regeneration, as evaluated by immunocytochemistry using a marker - ß-tubulin. The dual bFGF/NGF-immobilized PCL/Pluronic F127 microspheres may be a promising candidate for nerve regeneration in certain target tissues (i.e. muscles) leading to sufficient reinnervation.

[Vegetative state treated with acupoint injection combined with plum-blossom needle in children: a randomized controlled trial].

OBJECTIVE: To observe the clinical efficacy difference on vegetative state in children between acupoint injection combined with plum-blossom needle and western medication based on basic treatment. METHODS: Forty-eight children of vegetative state were randomized into an observation group and a control group, 24 cases in each one. On the basis of the treatment of transcranial magnetic stimulation apparatus, balancing treatment apparatus and massage, the acupoint injection and tapping method with plum-blossom needle were adopted in the observation group, in which Xingnaojing injection, mouse nerve growth factor (mNGF) injection, monosialotetrahexosylganglioside sodium injection (MSI), compound Danshen injection were divided in 6 pairs and were injected respectively in Baihui (GV 20), Yongquan (KI 1), Fengfu (GV 16), Yamen (GV 15) and the others, 0.5 mL in each acupoint, once a day for continuous 10 days. Additionally, the tapping method with plum-blossom needle was used on the Governor Vessel and Jiaji (EX-B 2) on the back. In the control group, the intravenous infusion was adopted with citicoline sodium injection, mannitol injection and dexamethasone injection. The treatment was given once a day, 20 days of treatment made one session and totally 3 sessions were required in the two groups. The clinical efficacy, the vegetative state score and the mean curing time were observed after 20 days, 40 days and 60 days of treatment between the two groups. RESULTS: The effective rates were 58.3% (14/24), 70.8% (17/24) and 79.2% (19/24) in 20 days, 40 days and 60 days of treatment in the observation group and 20.8% (5/24), 45.8% (11/24) and 58.3% (14/24) in the control group respectively. The efficacy in the observation group was superior to those in the control group (P < 0.01, P < 0.05). The vegetative state score was improved apparently after 20 days, 40 days and 60 days of treatment as compared with those before treatment separately (all P < 0.05). It was improved obviously at the each time point after treatment in the observation group as compared with that in the control group (3.34 +/- 2.41 vs 2.64 +/- 11.56, 6.20 +/- 1.46 vs 4.34 +/- 1.64, 11.26 +/- 2.63 vs 8.75 +/- 2.18, all P < 0.05). The mean curing time was (45.67 +/- 16.24) days in the observation group, which was shorter apparently than that of (55.34 +/- 4.57) days in the control group (P < 0.05). CONCLUSION: Based on basic treatment acupoint injection combined with tapping method of plum-blossom needle achieve the reliable efficacy on vegetative state in children.

Assembly of protein-based hollow spheres encapsulating a therapeutic factor.

Neurotrophins, as important regulators of neural development, function, and survival, have a therapeutic potential to repair damaged neurons. However, a controlled delivery of therapeutic molecules to injured tissue remains one of the greatest challenges facing the translation of novel drug therapeutics field. This study presents the development of an innovative protein-protein delivery technology of nerve growth factor (NGF) by an electrostatically assembled protein-based (collagen) reservoir system that can be directly injected into the injury site and provide long-term release of the therapeutic. A protein-based biomimetic hollow reservoir system was fabricated using a template method. The capability of neurotrophins to localize in these reservoir systems was confirmed by confocal images of fluorescently labeled collagen and NGF. In addition, high loading efficiency of the reservoir system was proven using ELISA. By comparing release profile from microspheres with varying cross-linking, highly cross-linked collagen spheres were chosen as they have the slowest release rate. Finally, biological activity of released NGF was assessed using rat pheochromocytoma (PC12) cell line and primary rat dorsal root ganglion (DRG) cell bioassay where cell treatment with NGF-loaded reservoirs induced significant neuronal outgrowth, similar to that seen in NGF treated controls. Data presented here highlights the potential of a high capacity reservoir-growth factor technology as a promising therapeutic treatment for neuroregenerative applications and other neurodegenerative diseases.

Effect of an adipose-derived stem cell and nerve growth factor-incorporated hydrogel on recovery of erectile function in a rat model of cavernous nerve injury.

Postprostatectomy erectile dysfunction (ED) is the major problem for patients with clinically localized prostate cancer. Recently, gene and stem cell-based therapy of the corpus cavernosum has been attempted for postprostatectomy ED, but those therapies are limited by rapid blood flow and disruption of the normal architecture of the corpus cavernosum. In this study, we attempted to regenerate the damaged cavernous nerve (CN), which is the main cause of ED. We investigated the effectiveness of human adipose-derived stem cell (hADSC) and nerve growth factor-incorporated hyaluronic acid-based hydrogel (NGF-hydrogel) application on the CN in a rat model of bilateral cavernous nerve crush injury. Four weeks after the operation, erectile function was assessed by detecting the intracavernous pressure (ICP)/arterial pressure level by CN electrostimulation. The ICP was significantly increased by application of hADSC with NGF-hydrogel compared to the other experimental groups. CN and penile tissue were collected for histological examination. PKH-26 labeled hADSC colocalized with beta III tubulin were shown in CN tissue sections. hADSC/NGF-hydrogel treatment prevented smooth muscle atrophy in the corpus cavernosum. In addition, the hADSC/NGF-hydrogel group showed increased endothelial nitric oxide synthase protein expression. This study suggests that application of hADSCs with NGF-hydrogel on the CN might be a promising treatment for postprostatectomy ED.

The NGF point-injection for treatment of the sound-perceiving nerve deafness and tinnitus in 68 cases.

OBJECTIVE: To observe the therapeutic effects of the point-injection with nerve growth factor (NGF) for the sound-perceiving nerve deafness and tinnitus. METHODS: The 140 cases in this series were randomly divided into a treatment group of 68 cases treated by NGF injection at the points of Yifeng (TE 17) and Wangu (GB 12), and a control group of 72 cases orally taking Xibiling and adenosine triphosphate (ATP) and intramuscular injection with VB1 and VB12. RESULTS: The total effective rate was 78.6% in the treatment group and 31.8% in the control group, with significant difference between the two groups (P<0.05). CONCLUSION: For treating nervous deafness and tinnitus, NGF point-injection may show good therapeutic effects, but inversely proportional to the illness course, age and the extent of hypoacusis.

Combination therapy with intranasal NGF and electroacupuncture enhanced cell proliferation and survival in rats after stroke.

OBJECTIVE: This work was designed to investigate the effects of the combination therapy with intranasal (IN) administration of nerve growth factor (NGF) and electroacupuncture (EA) on neural progenitors and neurological functional recovery in adult rats after focal ischemia. METHODS: Rats subjected to 2 hours of middle cerebral artery occlusion (MCAO) were randomly assigned to four groups: Group 1, IN administration of phosphate-buffered saline (PBS) for control; Group 2, IN administration of NGF alone; Group 3, EA with IN administration of PBS; Group 4, IN administration of NGF with EA. Treatments were initiated at 2 hours after MCAO and continued for three consecutive days. All animals received daily injections of 5-bromodeoxyuridine (BrdU) intraperitoneally for 7 days starting at 24 hours after reperfusion and were killed at 2 hours or 21 days after the last BrdU injection. Neurological function and infarct volume were evaluated. Immunohistochemistry for BrdU was performed to identify newborn cells in the ipsilateral subventricular zone and striatum. RESULTS: The combination treatment led to significant improvement in neurological function and reduction in infarct volume. Cell proliferation and survival of progenitors were enhanced in rats treated with the combination treatment. CONCLUSION: These results suggest that IN administration of NGF and EA may have a synergistic effect in preventing ischemic injury and enhancing functional recovery after focal cerebral ischemia, which may be attributed to enhanced cell proliferation and survival.

Nasal administration of cholera toxin B subunit-nerve growth factor improves the space learning and memory abilities in beta-amyloid protein(25-35)-induced amnesic mice.

Nerve growth factor (NGF) is a potential drug for Alzheimer's disease treatment, but delivering NGF to the brain is difficult. To increase the content of NGF in brain, we prepared cholera toxin B subunit (CB) -NGF by the improved sodium metaperiodate method and compared its pharmacodynamics with NGF. In vitro, CB-NGF, as well as NGF, could promote neurite outgrowth and increase choline acetyltransferase activities. But the time window of TrkA phosphorylation induced by CB-NGF and NGF was different. In vivo, nasal administration of CB-NGF could increase the stay time and partially improve abilities of space learning and memory in amnesic mice, and protected the cholinergic neurons in basal forebrain against Abeta(25-35). CB-NGF treatment has better curative effects than NGF in Alzheimer's disease model mice.

Repeated restraint and nerve growth factor administration in male and female mice: effect on sympathetic and cardiovascular mediators of the stress response.

Chronic stress and increased sympathetic nerve activity have been associated with cardiovascular disorders such as hypertension, myocardial infarction and stroke. The aim of this study was to investigate the role of nerve growth factor (NGF) on the expression of tyrosine hydroxylase (TH), vascular-endothelial growth factor (VEGF) and leptin receptor (OB-R) in brain, adrenal and cardiovascular tissues of adult male and female mice following a chronic stress procedure. It was found that daily restraint for 10 consecutive days alters TH levels in hypothalamic and brainstem areas related to sympathetic activation, in both male and female mice. Chronic stress procedure also modifies heart and aorta VEGF levels in male mice, and adrenal glands TH in female mice. The NGF administration in stressed mice reverted the stress-induced up-regulation of TH levels in male and female mice hypothalamic nuclei and in male locus coeruleus. Administration of NGF in stressed animals also down-regulated OB-R levels in the hypothalamus of both male and female mice and in the female aorta. Our findings indicate that repeated restraint in mice has an effect on TH and VEGF protein content at different brain and peripheral sites involved in the sympathetic and cardio-vascular response to stressful stimuli. NGF administration is able to counteract some of these stress-induced changes. Since NGF is known to be up-regulated during stress, a possible functional significance of our observations is that the circulating NGF released during and following stress may serve to prevent possible deficits and/or damage linked to stress-induced sympathetic and cardiovascular activation.

Brainstem and cervical spinal cord Fos immunoreactivity evoked by nerve growth factor injection into neck muscles in mice.

Although myofascial tenderness is thought to play a key role in the pathophysiology of tension-type headache, very few studies have addressed neck muscle nociception. The neuronal activation pattern following local nerve growth factor (NGF) administration into semispinal neck muscles in anaesthetized mice was investigated using Fos protein immunohistochemistry. In order to differentiate between the effects of NGF administration on c-fos expression and the effects of surgical preparation, needle insertion and intramuscular injection, the experiments were conducted in three groups. In the sham group (n=7) cannula needles were only inserted without any injection. In the saline (n=7) and NGF groups (n=7) 0.9% physiological saline solution or 0.8 microm NGF solution were injected in both muscles, respectively. In comparison with sham and saline conditions, NGF administration induced significantly stronger Fos immunoreactivity in the mesencephalic periaqueductal grey (PAG), the medullary lateral reticular nucleus (LRN), and superficial layers I and II of cervical spinal dorsal horns C1, C2 and C3. This activation pattern corresponds very well to central nervous system processing of deep noxious input. A knowledge of the central anatomical representation of neck muscle pain is an essential prerequisite for the investigation of neck muscle nociception in order to develop a future model of tension-type headache.

MAK-5 treatment enhances the nerve growth factor-mediated neurite outgrowth in PC12 cells.

The effects of an ayurvedic compound (MAK-5) alone or together with nerve growth factor (NGF) on the neurite outgrowth of PC12 cells was studied. PC12 cells treated with NGF alone showed a clear neurite outgrowth with a decrease of the proliferation at the dose higher than 5 ng/ml. MAK-5 alone does not induce significant neurite outgrowth in the PC12 cells and does not decrease the proliferation. The PC12 cells treated with NGF supplemented with MAK-5 showed a well-evident morphological differentiation also at low doses of NGF (less than 5 ng/ml), however, the proliferation does not decrease. We suggest that MAK-5 could contain some differentiating agents that are able to potentiate NGF inducing neuronal differentiation in PC12 cells without decreasing the cell proliferation.

Directed and enhanced neurite growth with pulsed magnetic field stimulation.

Pulsed magnetic field (PMF) stimulation was applied to mammalian neurons in vitro to influence axonal growth and to determine whether induced current would direct and enhance neurite growth in the direction of the current. Two coils were constructed from individual sheets of copper folded into a square coil. Each coil was placed in a separate water-jacketed incubator. One was energized by a waveform generator driving a power amplifier, the other was not energized. Whole dorsal root ganglia (DRG) explant cultures from 15-day Sprague-Dawley rat embryos were established in supplemented media plus nerve growth factor (NGF) at concentrations of 0-100 ng/mL on a collagen-laminin substrate. Dishes were placed at the center of the top and bottom of both coils, so that the DRG were adjacent to the current flowing in the coil. After an initial 12 h allowing DRG attachment to the substrate floor, one coil was energized for 18 h, followed by a postexposure period of 18 h. Total incubation time was 48 h for all DRG cultures. At termination, DRG were histochemically stained for visualization and quantitative analysis of neurite outgrowth. Direction and length of neurite outgrowth were recorded with respect to direction of the current. PMF exposed DRG exhibited asymmetrical growth parallel to the current direction with concomitant enhancement of neurite length. DRG cultures not PMF exposed had a characteristic radial pattern of neurite outgrowth. These results suggest that PMF may offer a noninvasive mechanism to direct and promote nerve regeneration.

NGF treatment potentiates c-fos expression in the rat nucleus basalis upon excitotoxic lesion with quisqualic acid.

The induction of the c-fos gene in the rat brain by NGF was studied in a model of acute cholinergic hypofunction, i.e., the lesion of the nucleus basalis magnocellularis (NBM) with quisqualic acid. Choline acetyltransferase and Fos immunoreactivity (IR) in the NBM were analyzed at different times after the excitotoxic lesion. NGF treatment induced a potentiation of Fos expression 4 and 24 h after lesion. The possibility is discussed that c-fos induction is one of the early mechanisms of the neuroprotective action of NGF.