Increased ciliary neurotrophic factor in blood serum and lacrimal fluid as a potential biomarkers of focal epilepsy.

PURPOSE: To evaluate ciliary neurotrophic factor (CNTF) level in blood serum (BS) and lacrimal fluid (LF) of people with epilepsy (PWE). METHODS: A case-control study of 72 consecutive patients with focal epilepsy (cases, epilepsy group) and 60 age- and gender-matched healthy volunteers (controls) was performed. Based on comorbid depression, two subgroups of PWE were formed. CNTF level was measured by an enzyme-linked immunosorbent assay (ELISA) in the BS and LF. For measurements of low CNTF levels in the BS, the methodology previously improved by the authors was applied. RESULTS: As compared to controls, CNTF level (pg/mL) in PWE was increased both in the BS (7.0±2.9 vs. 3.7±2.0, P<0.000) and in LF (34.0±8.0 vs. 30.6±4.8, P=0.005). No significant correlation was found between CNTF level in the BS and LF either in PWE or in controls. No impact of comorbid depression or any demographic or clinical parameters studied on CNTF level in the BS or LF of PWE could be detected. CONCLUSIONS: In patients with focal epilepsy, CNTF level is increased both in the BS and LF, though without correlation between them. No association of CNTF levels with age, gender, or clinical parameters, as well as depression occurrence, was found. High CNTF levels in the BS and LF could be considered as non-invasive biomarkers of focal epilepsy.

Therapeutic potential of conditioned medium derived from oligodendrocytes cultured in a self-assembling peptide nanoscaffold in experimental autoimmune encephalomyelitis.

The use of neurotrophic factors is considered to be a novel therapeutic approach for restoring and/or maintaining neurological function in neurodegenerative disorders, such as multiple sclerosis (MS). Various studies have shown that conditioned medium produced by oligodendrocyte (OL-CM) contain a variety of neurotrophic factors. Here, we investigated the restorative effects of OL-CM, collected from oligodendrocytes cultured in a self-assembling peptide hydrogels scaffold (PuraMatrix), in experimental autoimmune encephalomyelitis (EAE) mouse model. Neural stem/progenitor cells, isolated from the embryonic mouse brain, were cultured and differentiated into oligodendrocyte. Cell viability and proliferation of oligodendrocytes were assessed by live/dead and MTT assays. Motor functions, myelination, cell infiltration, gliosis, and inflammatory process were assessed in EAE mice after intracranial injection of OL-CM at different concentrations. Application of OL-CM improved clinical score and neurological function in EAE mice and reduced the inflammatory cell infiltration and demyelination. Furthermore, administration of OL-CM reduced the expression of pro-inflammatory cytokines and suppressed the activation of NLRP3-inflammasome complex in EAE mice. These data suggest the potential therapeutic effect of OL-CM for MS treatment.

Microglia support ATF3-positive neurons following hypoglossal nerve axotomy.

Microglia are essential in developmental processes and maintenance of neuronal homeostasis. Experimental axotomy of motor neurons results in neurodegeneration, and microglia in motor nuclei become activated and migrate towards injured neurons. However, whether these activated microglia are protective or destructive to neurons remains controversial. In the present study, we transected the hypoglossal nerve in BALB/c mice, causing activating transcription factor 3 (ATF3) and growth associated protein 43 (GAP43) induction, and partial neuronal death. Inhibition of microglial accumulation by minocycline administration impaired microglial accumulation, decreased GAP43 mRNA expression, and reduced motor neuron survival. Expression of ATF3 contributed to nerve regeneration, and increased within 6 h after axotomy, prior to microglial migration. Further, microglial contact with neuronal cell bodies was associated with neuronal ATF3 expression. Colchicine administration blocked lesion-induced ATF3 transcription in axotomized neurons and microglial accumulation. In addition, perineuronal microglia-derived ciliary neurotrophic factor (CNTF) increased, indicating that perineuronal microglia in the hypoglossal nucleus protect axotomized motor neurons by releasing trophic factors. We also observed that microglia secrete CNTF and that neurons have CNTFRα and can respond to it in vitro. CNTF promote neurite elongation and neuronal survival of primary cultured neurons. Microglia make contact through unknown neuronal signals that are possibly regulated by ATF3 in hypoglossal nucleus. Moreover, they play important roles in regenerating motor neurons and are potential new therapeutic targets for motor neuron diseases.

Prolonged Jaw Opening Promotes Nociception and Enhanced Cytokine Expression.

AIMS: To test the hypothesis that prolonged jaw opening, as can occur during routine dental procedures, increases nociceptive sensitivity of the masseter muscle and increases cytokine expression. METHODS: Sprague-Dawley rats were used to investigate behavioral and cellular changes in response to prolonged jaw opening. A surgical retractor was placed around the maxillary and mandibular incisors, and the jaw was held at near maximal opening for 20 minutes. Head-withdrawal responses to mechanical stimuli applied to the facial skin overlying the left and right masseter muscles were determined following jaw opening. Cytokine levels in the upper cervical spinal cord containing the caudal part of the spinal trigeminal nucleus were evaluated using protein antibody microarrays (n = 3). Statistical analysis was performed using a nonparametric Mann-Whitney U test. RESULTS: Prolonged jaw opening significantly increased nocifensive head withdrawal to mechanical stimuli at 2 hours, and days 3 and 7 postinduction (P < .05). The increase in nociceptive response resolved after 14 days. Sustained jaw opening also stimulated differential cytokine expression in the trigeminal ganglion and upper cervical spinal cord that persisted 14 days postprocedure (P < .05). CONCLUSION: These findings provide evidence that near maximal jaw opening can lead to activation and prolonged sensitization of trigeminal neurons that results in nociceptive behavior evoked by stimulation of the masseter muscle, a physiologic event often associated with temporomandibular disorders (TMD). Results from this study may provide a plausible explanation for why some patients develop TMD after routine dental procedures that involve prolonged jaw opening.

Loss of PEG chain in routine SDS-PAGE analysis of PEG-maleimide modified protein.

SDS-PAGE represents a quick and simple method for qualitative and quantitative analysis of protein and protein-containing conjugates, mostly pegylated proteins. PEG-maleimide (MAL) is frequently used to site-specifically pegylate therapeutic proteins via free cysteine residue by forming a thiosuccinimide structure for pursuing homogeneous products. The C-S linkage between protein and PEG-MAL is generally thought to be relatively stable. However, loss of intact PEG chain in routine SDS-PAGE analysis of PEG-maleimide modified protein was observed. It is a thiol-independent thioether cleavage and the shedding of PEG chain exclusively happens to PEG-MAL modified conjugates although PEG-vinylsulfone conjugates to thiol-containing proteins also through a C-S linkage. Cleavage kinetics of PEG40k-MAL modified ciliary neurotrophic factor showed this kind of degradation could immediately happen even in 1 min incubation at high temperature and could be detected at physiological temperature and pH, although the rate was relatively slow. This may provide another degradation route for maleimide-thiol conjugate irrespective of reactive thiol, although the specific mechanism is still not very clear for us. It would also offer a basis for accurate characterization of PEG-MAL modified protein/peptide by SDS-PAGE analysis.

[Effects of bone marrow mesenchymal stem cell transplantation on retinal cell apoptosis in premature rats with retinopathy].

OBJECTIVE: To explore the effects of marrow mesenchymal stem cell (BMSC) transplantation on retinal cells apoptosis and changes to neurotrophin-3 (NT-3 and ciliary neurotrophic factor (CNTF) in rats with retinopathy of prematurity (ROP). METHODS: Seven-day-old Sprague-Dawley rats were randomly divided into normal control (CON), ROP, BMSC transplantation (BMSCs were transplanted 5 days after oxygen conditioning) and phosphate buffered saline (PBS) groups. The ROP model was prepared according to the classic hyperoxygen method. Seven days after transplantation, TUNEL/DAPI, NT-3/API and CNTF/DAPI double-labeled immunofluorescence were used to examine the effects of BMSC transplantation on both the apoptosis of retinal cells and the expression of NT-3 and CNTF protein in the retinal cells of the ROP rats. RESULTS: Seven days after BMSC transplantation, there were few TUNEL+ DAPI+ cells observed in the CON group. There were fewer TUNEL+DAPI+ cells observed in the BMSC group than in the ROP group (P<0.01), but there was no significant difference between the ROP and PBS groups (P>0.05). There were few NT-3+DAPI+ cells and CNTF+DAPI+ cells in the CON group. There were more NT-3+DAPI+ and CNTF+DAPI+ cells in the ROP group than in the CON group, but there was no significant difference between the ROP and CON groups (P>0.05). More NT-3+DAPI+ and CNTF+DAPI+ cells were observed in the BMSC group compared with the ROP group (P<0.01), and there was no significant difference in either NT-3+DAPI+ or CNTF+DAPI+ cells between the ROP and PBS groups (P>0.05). CONCLUSIONS: BMSC transplantation therapy could alleviate the apoptosis of retinal cells in ROP rats, and its mechanisms might be associated with promoting the expression of NT-3 and CNTF protein in retinal cells.

Expression of ciliary neurotrophic factor after induction of ocular hypertension in the retina of rats.

BACKGROUND: Glaucoma is mainly characterized by the loss of retinal ganglion cells. Ciliary neurotrophic factor (CNTF) is believed to stimulate the regeneration of axons of retinal ganglion cells. The objective of our study was to detect the expression of CNTF in the retina of a rat glaucoma model with increased intraocular pressure (IOP). METHODS: The rat glaucoma model was set up by electrocoagulating at least three episcleral and limbal veins. The location and the expression level of CNTF were detected at 1, 3, 7, 14, and 28 days post-surgery by immunohistochemistry, semiquantitative reverse-transcription polymerase chain reaction (RT-PCR), and Western blot analysis. RESULTS: The rat glaucoma model with chronic, moderately elevated IOP was successfully produced. A minimum expression of CNTF was found in the ganglion cell layer of the retinas of the control group, and temporally increased expression and intensity of CNTF were found in the experimental retinas. CONCLUSION: The expression of endogenous CNTF in the rat retina was found altered after the induction of ocular hypertension.

Ciliary neurotrophic factor (CNTF) in the olfactory system of rats and mice.

The rodent olfactory system is a regarded model for the relationship between neurotrophic factors, their receptors, and their compound influence on the notable lifelong neuroplasticity occurring in this sensory system. It was known that high amounts of ciliary neurotrophic factor (CNTF), a hematopoietic cytokine, can be found in the olfactory bulb. In the awarded work, a detailed cellular characterization of CNTF-localization in the olfactory system was obtained. The results demonstrated CNTF-immunoreactivity in olfactory ensheathing cells, newborn interneurons in the olfactory bulb, and in a subpopulation of mature olfactory sensory neurons in the olfactory epithelium. Three-dimensional reconstructions of CNTF-immunoreactive axonal bulbar projections of these neurons revealed an ordered bilaterally symmetric pattern. This finding implies a potential connection between neuronal CNTF-expression in the olfactory epithelium and olfactory information processing.

AAV-mediated expression of CNTF promotes long-term survival and regeneration of adult rat retinal ganglion cells.

We compared the effects of intravitreal injection of bi-cistronic adeno-associated viral (AAV-2) vectors encoding enhanced green fluorescent protein (GFP) and either ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF) or growth-associated protein-43 (GAP43) on adult retinal ganglion cell (RGC) survival and regeneration following (i) optic nerve (ON) crush or (ii) after ON cut and attachment of a peripheral nerve (PN). At 7 weeks after ON crush, quantification of betaIII-tubulin immunostaining revealed that, compared to AAV-GFP controls, RGC survival was not enhanced by AAV-GAP43-GFP but was increased in AAV-CNTF-GFP (mean RGCs/retina: 17 450+/-358 s.e.m.) and AAV-BDNF-GFP injected eyes (10 200+/-4064 RGCs/retina). Consistent with increased RGC viability in AAV-CNTF-GFP and AAV-BDNF-GFP injected eyes, these animals possessed many betaIII-tubulin- and GFP-positive fibres proximal to the ON crush. However, only in the AAV-CNTF-GFP group were regenerating RGC axons seen in distal ON (1135+/-367 axons/nerve, 0.5 mm post-crush), some reaching the optic chiasm. RGCs were immunoreactive for CNTF and quantitative RT-PCR revealed a substantial increase in CNTF mRNA expression in retinas transduced with AAV-CNTF-GFP. The combination of AAV-CNTF-GFP transduction of RGCs with autologous PN-ON transplantation resulted in even greater RGC survival and regeneration. At 7 weeks after PN transplantation there were 27 954 (+/-2833) surviving RGCs/retina, about 25% of the adult RGC population. Of these, 13 352 (+/-1868) RGCs/retina were retrogradely labelled after fluorogold injections into PN grafts. In summary, AAV-mediated expression of CNTF promotes long-term survival and regeneration of injured adult RGCs, effects that are substantially enhanced by combining gene and cell-based therapies/interventions.

Lentiviral-mediated transfer of CNTF to schwann cells within reconstructed peripheral nerve grafts enhances adult retinal ganglion cell survival and axonal regeneration.

We recently described a method for reconstituting peripheral nerve (PN) sheaths using adult Schwann cells (SCs). Reconstructed PN tissue grafted onto the cut optic nerve supports the regeneration of injured adult rat retinal ganglion cell (RGC) axons. To determine whether genetic manipulation of such grafts can further enhance regeneration, adult SCs were transduced with lentiviral vectors encoding either ciliary neurotrophic factor (LV-CNTF) or green fluorescent protein (LV-GFP). SCs expressed transgenes for at least 4 weeks after transplantation. There were high levels of CNTF mRNA and CNTF protein in PN grafts containing LV-CNTF-transduced SCs. Mean RGC survival was significantly increased with these grafts (11,863/retina) compared with LV-GFP controls (7064/retina). LV-CNTF-transduced SCs enhanced axonal regeneration to an even greater extent (3097 vs 393 RGCs/retina in LV-GFP controls). Many regenerated axons were myelinated. The use of genetically modified, reconstituted PN grafts to bridge tissue defects may provide new therapeutic strategies for the treatment of both CNS and PNS injuries.

Increased expression of IL-6 and LIF in the hypertrophied left ventricle of TGR(mRen2)27 and SHR rats.

Cytokines from the interleukin-6 (IL-6) family have been reported to play an important synergistic role with angiotensin II in the development of pathological cardiac hypertrophy. Whether their expression pattern changes in vivo, in an angiotensin I-dependent hypertrophied myocardium has not been reported. In this study, we addressed that issue using two animal models of angiotensin II-dependent cardiac hypertrophy. Heterozygous transgenic TGR(mRen2)27 (TGR) with an overactive cardiac renin angiotensin system and the closely related spontaneously hypertensive rats (SHR) were compared to their respective control rats. The mRNA levels of IL-6, leukemia inhibitory factor (LIF), ciliary neurotrophic factor (CNTF) and cardiotrophin-1 (CT-1) as well as their receptor subunits, glycoprotein 130 (gp130), IL-6 receptor (IL-6R), LIFR, and CNTFR, were measured by semi-quantitative RT-PCR. The protein levels of IL-6, LIF and CT-1 were investigated by western blot. TGR and SHR both displayed significant over expression of mRNA and protein levels for IL-6 and LIF. In TGR, the increased level of LIF was accompanied by a decrease in mRNA levels for LIFR and CNTFR. In SHR, a higher level of mRNA IL-6R was observed. By contrast, the mRNA and protein levels for CT-1 and the mRNA level for gp130 did not vary in these two models. These findings suggest that IL-6 and LIF, but not CT-1, contribute to angiotensin II-dependent left ventricular hypertrophy in the two hypertensive rat models, TGR(mRen2)27 and SHR.

Quinolinic acid lesions of the caudate putamen in the rat lead to a local increase of ciliary neurotrophic factor.

When applied prior to excitotoxic lesions, ciliary neurotrophic factor (CNTF) has been shown to be neuroprotective. However, data concerning the endogenous CNTF content of the intact rat striatum are rare and have not until now been available for the quinolinic acid (QA)-lesioned striatum. Therefore, we investigated the CNTF content in the QA-lesioned rat striatum for at least 1 month using immunohistochemistry and Western blot analysis. In lesioned striata a neuronal loss was observed by Nissl staining and by a reduction of NeuN-immunoreactive cells, whereas increased glial fibrillary acidic protein immunoreactivity showed a gliotic reaction. With CNTF immunohistochemistry we found that in the QA-lesioned striatum CNTF was increased over time, whereas it was not detectable in intact and sham-lesioned striata. CNTF-immunoreactive cells had the morphology of protoplasmatic astrocytes. Furthermore, quantitative Western blotting demonstrated that the content of CNTF protein from striatal lysates containing 1 mg of whole protein 1 month after QA lesioning (2.76 +/- 1.71 ng) was significantly increased (P < 0.05, U-test) compared with sham-lesioned hemispheres (0.68 +/- 0.25 ng) and intact controls (0.55 +/- 0.25 ng). We conclude that CNTF content is correlated with glial scar formation and suggest that our results may be of relevance to cell grafting strategies for the treatment of Huntington's disease.

Peritoneal fluid concentrations of ciliary neurotrophic factor, a gp130 cytokine, in women with endometriosis.

Ciliary neurotrophic factor (CNTF) is a gp130 neuroregulatory cytokine belonging to the interleukin-6-type cytokine superfamily. Several lines of evidence suggest that the concentrations of interleukin-6 are elevated in the peritoneal fluid of endometriosis patients. To our knowledge, no study on whether this might also be the case for CNTF levels has been published previously. The CNTF concentrations in the peritoneal fluid were measured by ELISA in 51 women with (n = 31) and without (n = 20) endometriosis. Surgery was scheduled during the proliferative or the secretory phase of the menstrual cycle. CNTF was detectable in the peritoneal fluid of 43% of the women tested. The concentrations of CNTF showed no correlation with the presence of endometriosis or the phase of the menstrual cycle. We found no evidence to suggest that CNTF is involved in the pathogenesis of pelvic endometriosis.

Ciliary neurotrophic factor in the olfactory bulb of rats and mice.

Ciliary neurotrophic factor (CNTF) is primarily regarded as an astrocytic lesion factor, promoting neuronal survival and influencing plasticity processes in deafferented areas of the CNS. Postnatal loss of neurons in CNTF-deficient mice indicates a function of the factor also under physiological conditions. In the olfactory bulb, where neurogenesis, axo- and synaptogenesis continue throughout life, CNTF content is constitutively high. The cellular localization of CNTF in the rat olfactory bulb is not fully resolved, and species differences between mouse and rat are not yet characterized. In the present study, four different CNTF antibodies and double immunolabeling with specific markers for glial and neuronal cells were used to study the cellular localization of CNTF in rat and mouse olfactory bulb. Specificity of the detection was checked with tissue from CNTF-deficient mice, and investigations were complemented by immunolocalization of reporter protein in mice synthesizing beta-galactosidase under control of the CNTF promoter (CNTF lacZ-knock-in mice). In both species, CNTF localized to ensheathing cell nuclei, cell bodies and axon-enveloping processes. Additionally, individual axons of olfactory neurons were CNTF immunoreactive. Both CNTF protein content and immunoreaction intensity were lower in mice than in rats. Scattered lightly CNTF-reactive cells were found in the granular and external plexiform layers in rats. Some CNTF-positive cells were associated with immunoreactivity for the polysialylated form of the neural cell adhesion molecule, which is expressed by maturing interneurons derived from the rostral migratory stream. In CNTF lacZ-knock-in mice, beta-galactosidase reactivity was found in ensheathing cells of the olfactory nerve layer, and in cells of the glomerular, external plexiform and granular layers. The study proves that CNTF is localized in glial and neuronal structures in the rodent olfactory bulb. Results in mice provide a basis for investigations concerning the effects of a lack of the factor in CNTF-deficient mice.

GFAP knockout mice have increased levels of GDNF that protect striatal neurons from metabolic and excitotoxic insults.

In response to injury and degeneration, astrocytes hypertrophy, extend processes, and increase production of glial fibrillary acidic protein (GFAP), an intermediate filament protein located within their cytoplasm. The present study tested the hypothesis that GFAP expression alters the vulnerability of neurons to excitotoxic and metabolic insult induced by 3-nitroproprionic acid (3-NP), an irreversible inhibitor of mitochondrial complex II activity or the excitotoxin quinolinic acid (QA). In this respect, adult GFAP knockout mice (KO) and wild-type control mice (WT) received unilateral intrastriatal injections of 3-NP (200 nmol/microl) or QA (100 nmol/microl) and were killed 1, 2, or 4 weeks later. Lesion volume and neuronal counts were quantified using unbiased stereologic principles. For both QA and 3-NP lesions, a significant decrease in lesion volume and an increase in striatal projection neurons were seen in GFAP KO mice compared with WT mice. Enzyme-linked immunoassay analysis revealed increased basal levels of glial cell derived neurotrophic factor (GDNF) relative to WT mice. In contrast, no differences were observed in the expression of ciliary neurotrophic factor or nerve growth factor. These data strongly suggest that the expression of GFAP is implicated with the production of GDNF to a degree that confers neuroprotection after an excitotoxic or metabolic insult.

Adenoviral gene transfer of GDNF, BDNF and TGF beta 2, but not CNTF, cardiotrophin-1 or IGF1, protects injured adult motoneurons after facial nerve avulsion.

We examined neuroprotective effects of recombinant adenoviral vectors encoding glial cell line-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), cardiotrophin-1 (CT1), insulin-like growth factor-1 (IGF1), and transforming growth factor-beta2 (TGFbeta2) on lesioned adult rat facial motoneurons. The right facial nerves of adult Fischer 344 male rats were avulsed and removed from the stylomastoid foramen, and adenoviral vectors were injected into the facial canal. Animals avulsed and treated with adenovirus encoding GDNF, BDNF, CNTF, CT1, IGF1 and TGFbeta2 showed intense immunolabeling for these factors in lesioned facial motoneurons, respectively, indicating adenoviral induction of the neurotrophic factors in these neurons. The treatment with adenovirus encoding GDNF, BDNF, or TGFbeta2 after avulsion significantly prevented the loss of lesioned facial motoneurons, improved choline acetyltransferase immunoreactivity and prevented the induction of nitric oxide synthase activity in these neurons. The treatment with adenovirus encoding CNTF, CT1 or IGF1, however, failed to protect these neurons after avulsion. These results indicate that the gene transfer of GDNF and BDNF and TGFbeta2 but not CNTF, CT1 or IGF1 may prevent the degeneration of motoneurons in adult humans with motoneuron injury and motor neuron diseases.

Immunofluorescence and biochemical techniques to detect nuclear localization of ciliary neurotrophic factor in glial cells.

Ciliary neurotrophic factor (CNTF) promotes the survival of several populations of neurons, including sensory and motor neurons. It is mainly produced by Schwann cells and astrocytes and exerts its biological function via a specific membrane receptor. We recently determined the nuclear localization of CNTF in producing cells, after transfection and in the heterologous system of Xenopus oocytes. In the present paper, we describe in detail the techniques for the detection of CNTF in the nucleus of rat astrocytes, transfected cells, isolated nuclei and injected Xenopus oocytes.

Neurotrophins and other growth factors in the regenerative milieu of proximal nerve stump tips.

Classic ideas on mechanisms for axon sprouting and nerve regeneration from peripheral nerves suggest that there is a prominent role for neurotrophin support. There has been comparatively less attention towards features of the regenerative process that develop from the proximal nerve trunk without the support of target tissues or the denervated trunk of a peripheral nerve. We studied early (2-14 d) expression of local growth factors in proximal nerve stump tips of transected sciatic nerves in rats. Immunohistochemical labelling was used to address specific deposition of BDNF, NGF, NT-3, bFGF, CNTF and IGF-1. We observed a unique localisation of BDNF, and to a much lesser extent, NGF in mast cells of injured nerve trunks but they were also observed in intact uninjured nerves. Macrophages did not express either BDNF or NGF. CNTF and IGF-1 were expressed in Schwann cells of intact nerves and stumps. We did not observe bFGF or NT-3 expression in any of the samples we studied. Mast cells may represent an important reservoir of BDNF in peripheral nerves.

Developmental expression of urinary bladder neurotrophic factor mRNA and protein in the neonatal rat.

These studies were performed to determine the developmental expression pattern of neurotrophic factor (NTF: nerve growth factor (betaNGF), brain-derived neurotrophic factor (BDNF), glial-derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF), neurotrophin-3 (NT-3) and NT-4 mRNA and NGF, NT-3 and NT-4 protein in the urinary bladder of the postnatal Wistar rat. It was hypothesized that NTFs may contribute to the development of the spinobulbospinal micturition reflex that represents the adult micturition pattern. Changes in NTF mRNA or protein expression in the urinary bladder at the time of development of the mature micturition reflex (postnatal days (P) 16-18) may suggest an involvement of target-derived NTFs in this maturation process. Developmental ages, prior to (P5, P10, P15) or following (P20, P30, adult P90) the development of the spinobulbospinal micturition reflex were selected and the urinary bladder was analyzed for levels of neurotrophic factor mRNA or protein. Results from ribonuclease protection assays demonstrated a similar developmental pattern among each neurotrophic factor examined. Neurotrophic factor mRNA levels increased by P10 and reach a maximum by P15. Subsequently, NTF mRNA levels declined to adult levels that were less than the earliest postnatal time examined (P5). NTF mRNA expression was significantly (p

Changes in urinary bladder neurotrophic factor mRNA and NGF protein following urinary bladder dysfunction.

Spinal cord injury and cyclophosphamide-induced cystitis dramatically alter lower urinary tract function and produce neurochemical, electrophysiological, and anatomical changes that may contribute to reorganization of the micturition reflex. Mechanisms underlying this neural plasticity may involve alterations in neurotrophic factors in the urinary bladder. These studies have determined neurotrophic factors in the urinary bladder that may contribute to reorganization of the micturition reflex following cystitis or spinal cord injury. A ribonuclease protection assay was used to measure changes in urinary bladder neurotrophic factor mRNA (betaNGF, BDNF, GDNF, CNTF, NT-3, and NT-4) following spinal cord injury (acute/chronic) or cyclophosphamide-induced cystitis (acute/chronic). The correlation between urinary bladder nerve growth factor mRNA and nerve growth factor protein expression was also determined. Each experimental paradigm resulted in significant (P