Lori G. Isaacson

Estrogen regulation of neurotrophin expression in sympathetic neurons and vascular targets

We hypothesize that estrogen exerts a modulatory effect on sympathetic neurons to reduce neural cardiovascular tone and that these effects are modulated by nerve growth factor (NGF), a neurotrophin that regulates sympathetic neuron survival and maintenance. We examined the effects of estrogen on NGF and tyrosine hydroxylase (TH) protein content in specific vascular targets. Ovariectomized, adult Sprague-Dawley rats were implanted with placebo or 17beta-estradiol (release rate, 0.05 mg/day). Fourteen days later, NGF levels in the superior cervical ganglia (SCG) and its targets, the heart, external carotid artery, and the extracerebral blood vessels, as well as estrogen receptor alpha (ERalpha) content levels in the heart, were determined using semi-quantitative Western blot analysis. TH levels in the SCG and extracerebral blood vessels were determined by Western blotting and immunocytochemistry, respectively. Circulating levels of 17beta-estradiol and prolactin (PRL) were quantified by RIA. Estrogen replacement significantly decreased NGF protein in the SCG and its targets, the external carotid artery, heart and extracerebral blood vessels. TH protein associated with the extracerebral blood vessels was also significantly decreased, but ERalpha levels were significantly increased in the heart following estrogen replacement. These results indicate that estrogen reduces NGF protein content in sympathetic vascular targets, which may lead to decreased sympathetic innervations to these targets, and therefore reduced sympathetic regulation. In addition, the estrogen-induced increase in ERalpha levels in the heart, a target tissue of the SCG, suggests that estrogen may sensitize the heart to further estrogen modulation, and possibly increase vasodilation of the coronary vasculature.

Regulation of NGF and NT-3 protein expression in peripheral targets by sympathetic input.

Nerve growth factor (NGF) and neurotrophin-3 (NT-3) are target-derived proteins that regulate innervating sympathetic neurons. Here, we used western blot analysis to investigate changes in NGF and NT-3 protein in several peripheral tissues following loss of sympathetic input. Following removal of the superior cervical ganglion (SCG), large molecular weight (MW) NGF species, including proNGF-A, were increased in distal intracranial SCG targets, such as pineal gland and extracerebral blood vessels (bv). Mature NGF was a minor species in these tissues and unchanged following sympathectomy. Large MW NGF species also were increased when sympathectomy was followed by in vivo NGF administration. Mature NT-3, which was abundant in controls, was significantly decreased in these targets following sympathetic denervation. The decrease in mature NT-3 was enhanced following NGF administration. The trigeminal ganglion, which provides sensory input to these targets, showed increased NGF, but decreased NT-3, in these treatments, demonstrating that decreased NT-3 at the targets did not result from enhanced NT-3 uptake. Unlike pineal gland and extracerebral bv, the external carotid artery, an extracranial proximal SCG target, showed no change in NGF following denervation, and mature NT-3 was significantly increased. Following NGF administration, NT-3 was significantly decreased. We provide evidence for sympathetic regulation of NGF and NT-3 in peripheral targets and that elevated NGF can depress NT-3. The differential response in distal and proximal adult targets is consistent with the idea that neurons innervating proximal and distal targets may serve different roles in regulating neurotrophin protein. In addition, we conclude that previous ELISA results showing increased NGF protein following sympathetic denervation may have resulted from increases in large MW species, rather than an increase in mature NGF.

Plasticity of aged perivascular axons following exogenous NGF: analysis of catecholamines

The present study investigated the atrophy of aged perivascular sympathetic axons and the response of these cerebrovascular neurons to the neurotrophin nerve growth factor (NGF). Using high performance liquid chromatography coupled with electrochemical detection (HPLC-ECD) to quantify catecholamines and immunohistochemical methods to quantify the density of TH immunoreactive fibers, we found a significant decrease in norepinephrine (NE) and TH in aged sympathetic axons. However, following in vivo administration of exogenous neurotrophin, aged neurons exhibited a robust response to NGF that was similar to the young adult, suggesting little decline in the capability of aged neurons to utilize exogenous neurotrophin. These results suggest that the age-related atrophy of aged sympathetic axons may result primarily from reduced availability of target-derived neurotrophin rather than from intrinsic alterations of neuronal function.

Uninjured aged sympathetic neurons sprout in response to exogenous NGF in vivo

The extent to which the loss of plasticity by aged neurons is due to changes in the neuronal environment or to a loss of growth potential of the neurons has not been determined. In previous studies we observed that young adult cerebrovascular axons undergo a sprouting response following a 2-week intracerebroventricular infusion of nerve growth factor (15 microg; NGF). The present study used electron microscopy to examine the innervation of the intradural segment of the internal carotid artery of the aged rat and to determine whether aged sympathetic perivascular axons would respond to in vivo infusion of NGF. Young adult and aged Fischer 344 female rats received a 2-week intracranial infusion of NGF (15 microg) or vehicle (VEH) and were perfused for electron microscopy. Although there was no change in the total number of perivascular axons associated in aged VEH when compared with young adult VEH, a significant reduction was observed in aged VEH when total axons and sympathetic axons were expressed per microm2 vascular wall, reflecting an age-related increase in blood vessel size. Following NGF infusion, aged sympathetic axons were significantly increased by 192% compared with aged VEH cases. These results suggest that there is a proportional reduction in sympathetic cerebrovascular neurons with aging but that they exhibit robust sprouting in response to an exogenous neurotrophin.

Evidence for reduced accumulation of exogenous neurotrophin by aged sympathetic neurons.

The present study investigated the potential for neurotrophin uptake by cerebrovascular axons and subsequent accumulation in the aged superior cervical ganglion (SCG) following a two week intracerebroventricular infusion of nerve growth factor (NGF). In the SCG from aged rats, NGF protein levels declined significantly compared with the SCG from young adult rats. Following NGF infusion, perivascular axons from both young adult and aged rats showed intense NGF immunostaining. In addition, significant increases in NGF protein were shown using enzyme-linked immunosorbent assay (ELISA) and in counts of NGF immunopositive cell bodies in the SCG when compared with age-matched controls. NGF accumulation in ganglia from aged rats, however, was significantly less when compared with ganglia from young adult rats. The results of the present study suggest that NGF protein is significantly reduced in aged ganglia with the neurons retaining some capacity to take up and transport exogenous neurotrophin. Even so, the potential for NGF accumulation is dramatically reduced in aged rats when compared with that of young adult rats. While previous results have shown robust NGF-induced neurotransmitter responses by sympathetic neurons from the aged animal, the present finding of reduced accumulation of NGF in aged sympathetic neurons suggests an age-related difference in the utilization or transport of NGF.

Sympathetic ingrowth to the trigeminal ganglion following intracerebroventricular infusion of nerve growth factor.

The objective of the present study was to examine the remodeling of uninjured sympathetic axons in the adult rat trigeminal ganglion following a 2-week in vivo intracerebroventricular infusion of NGF. The accumulation of infused NGF in the trigeminal was assessed using ELISA and sympathetic fibers were localized immunohistochemically with an antibody to tyrosine hydroxylase (TH). In addition, high performance liquid chromatography coupled with electrochemical detection (HPLC-ECD) allowed for biochemical measurements of the catecholamines norepinephrine (NE) and dopamine (DA). Increased NGF protein in the trigeminal ganglion was paralleled by a significant increase in sympathetic fibers and pericellular plexuses (i.e. baskets) in the cell body regions. Some ganglia showed elevated NE following NGF infusion, yet the 88% increase in mean NE did not reach significance. Following bilateral removal of the sympathetic superior cervical ganglia (SCG), a significant reduction was observed in overall NE levels and in TH-immunoreactive (-ir) fibers in the cell body regions and peripheral branches, suggesting the SCG as the origin of the sympathetic ingrowth. However, mean DA levels as well as TH-ir fibers within the trigeminal central branch were unaffected by NGF infusion or removal of the SCG and likely resulted from intrinsic dopaminergic cell bodies. In conclusion, our data provide evidence that the increased availability of NGF in the young adult rat trigeminal ganglion observed following in vivo NGF infusion enhanced sympathetic associations with the sensory neurons in the trigeminal, supporting a role for NGF in the regulation of sympathosensory interactions.

Transection of preganglionic axons leads to CNS neuronal plasticity followed by survival and target reinnervation

The goals of the present study were to investigate the changes in sympathetic preganglionic neurons following transection of distal axons in the cervical sympathetic trunk (CST) that innervate the superior cervical ganglion (SCG) and to assess changes in the protein expression of brain derived neurotrophic factor (BDNF) and its receptor TrkB in the thoracic spinal cord. At 1 week, a significant decrease in soma volume and reduced soma expression of choline acetyltransferase (ChAT) in the intermediolateral cell column (IML) of T1 spinal cord were observed, with both ChAT-ir and non-immunoreactive neurons expressing the injury marker activating transcription factor 3. These changes were transient, and at later time points, ChAT expression and soma volume returned to control values and the number of ATF3 neurons declined. No evidence for cell loss or neuronal apoptosis was detected at any time point. Protein levels of BDNF and/or full length TrkB in the spinal cord were increased throughout the survival period. In the SCG, both ChAT-ir axons and ChAT protein remained decreased at 16 weeks, but were increased compared to the 10 week time point. These results suggest that though IML neurons show reduced ChAT expression and cell volume at 1 week following CST transection, at later time points, the neurons recovered and exhibited no significant signs of neurodegeneration. The alterations in BDNF and/or TrkB may have contributed to the survival of the IML neurons and the recovery of ChAT expression, as well as to the reinnervation of the SCG.

Characterization of trkB immunoreactive cells in the intermediolateral cell column of the rat spinal cord

The objective of the present study was to characterize the trkB receptor immunoreactive (-ir) cells in the intermediolateral cell column (IML) of the upper thoracic spinal cord. Small trkB-ir cells (area=56.1+/-4.4 microm(2)) observed in the IML showed characteristics of oligodendrocytes and were frequently observed in close apposition to choline acetyltransferase (ChAT)-ir cell bodies. Large trkB-ir cells (area=209.3+/-25.2 microm(2)) showed immunoreactivity for the neuronal marker NeuN, indicating their neuronal phenotype, as well as for ChAT, a marker for preganglionic neurons. TrkB and ChAT were co-localized in IML neurons primarily in cases that had received in vivo administration of nerve growth factor (NGF). These findings reveal two different cell types, oligodendrocytes and neurons, in the IML of the spinal cord that show trkB immunoreactivity, suggesting their regulation by brain derived neurotrophic factor (BDNF) and/or neurotrophin-4 (NT-4). In addition, there is evidence that NGF may play a role in the regulation of trkB-ir preganglionic neurons in the IML.

Sympathetic reinnervation of peripheral targets following bilateral axotomy of the adult superior cervical ganglion

The ability of adult injured postganglionic axons to reinnervate cerebrovascular targets is unknown, yet these axons can influence cerebral blood flow, particularly during REM sleep. The objective of the present study was to assess quantitatively the sympathetic reinnervation of vascular as well as non-vascular targets following bilateral axotomy of the superior cervical ganglion (SCG) at short term (1 day, 7 day) and long term (8 weeks, 12 weeks) survival time points. The sympathetic innervation of representative extracerebral blood vessels [internal carotid artery (ICA), basilar artery (BA), middle cerebral artery (MCA)], the submandibular gland (SMG), and pineal gland was quantified following injury using an antibody to tyrosine hydroxylase (TH). Changes in TH innervation were related to TH protein content in the SCG. At 7 day following bilateral SCG axotomy, all targets were significantly depleted of TH innervation, and the exact site on the BA where SCG input was lost could be discerned. Complete sympathetic reinnervation of the ICA was observed at long term survival times, yet TH innervation of other vascular targets showed significant decreases even at 12 weeks following axotomy. The SMG was fully reinnervated by 12 weeks, yet TH innervation of the pineal gland remained significantly decreased. TH protein in the SCG was significantly decreased at both short term and long term time points and showed little evidence of recovery. Our data demonstrate a slow reinnervation of most vascular targets following axotomy of the SCG with only minimal recovery of TH protein in the SCG at 12 weeks following injury.

Changes in NGF and NT-3 protein species in the superior cervical ganglion following axotomy of postganglionic axons.

Mature sympathetic neurons in the superior cervical ganglion (SCG) are regulated by target-derived neurotrophins such as nerve growth factor (NGF) and neurotrophin-3 (NT-3). High molecular weight NGF species and mature NT-3 are the predominant NGF and NT-3 protein isoforms in the SCG, yet it is unknown whether the presence of these species is dependent on intact connection with the target tissues. In an attempt to determine the role of peripheral targets in regulating the neurotrophin species found in the SCG, we investigated the NGF and NT-3 protein species present in the SCG following axotomy (transection) or injury of the post-ganglionic axons. Following a 7 day axotomy, the 22-24 kDa NGF species and the mature 14 kDa NT-3 species in the SCG were significantly reduced by 99% and 66% respectively, suggesting that intact connection with the target is necessary for the expression of these protein species. As expected, tyrosine hydroxylase (TH) protein in the SCG was significantly reduced by 80% at 7 days following axotomy. In order to distinguish between the effects of injury and loss of target connectivity, the SCG was examined following compression injury to the post-ganglionic nerves. Following injury, no reduction in the 22-24 kDa NGF or 14 kDa mature NT-3 species was observed in the SCG. TH protein was slightly, yet significantly, decreased in the SCG following injury. The findings of this study suggest that the presence of the 22-24 kDa NGF and mature 14 kDa NT-3 species in the SCG is dependent on connection with peripheral targets and may influence, at least in part, TH protein expression in adult sympathetic neurons.