Activated Microglia in the Rat Spinal Cord Following Peripheral Axon Injury Promote Glial and Neuronal Plasticity Which is Necessary for Long-Term Neuronal Survival.

Following the transection of peripheral sympathetic preganglionic axons comprising the cervical sympathetic trunk (CST), we observe robust glial and neuronal plasticity at 1 week post-injury in the rat spinal cord intermediolateral cell column (IML), which houses the injured parent neuronal cell bodies. This plasticity contributes to neuroprotection, as no neuronal loss in the IML is present at 16 weeks post-injury. Here, we administered the antibiotic minocycline or vehicle (VEH) daily for 1 week after CST transection to investigate the role of activated microglia in IML glial and neuronal plasticity and subsequent neuronal survival. At 1 week post-injury, minocycline treatment did not alter microglia number in the IML, but led to a dampened microglia activation state. In addition, the increases in oligodendrocyte (OL) lineage cells and activated astrocytes following injury in VEH rats were attenuated in the minocycline-treated rats. Further, the normal downregulation of choline acetyltransferase (ChAT) in the injured neurons was blunted. At 16 weeks post-injury, fewer ChAT+ neurons were present in the minocycline-treated rats, suggesting that activated microglia together with the glial and neuronal plasticity at 1 week post-injury contribute to the long-term survival of the injured neurons. These results provide evidence for beneficial crosstalk between activated microglia and neurons as well as other glial cells in the cord following peripheral axon injury, which ultimately leads to neuroprotection. The influences of microglia activation in promoting neuronal survival should be considered when developing therapies to administer minocycline for the treatment of neurological pathologies.

Elimination of microglia in mouse spinal cord alters the retrograde CNS plasticity observed following peripheral axon injury.

Following the transection of peripherally located sympathetic preganglionic axons of the cervical sympathetic trunk (CST), transient retrograde neuronal and glial responses occur in the intermediolateral cell column (IML) of the spinal cord, the location of the parent neuronal cell bodies. The role of microglia in this central response to peripheral axon injury was examined in mice fed the PLX5622 diet containing colony-stimulating factor-1 receptor (CSF-1R) inhibitor for 28 days, which eliminated approximately 90% of spinal cord microglia. Microglia elimination did not impact baseline neurotransmitter expression in the IML neurons, and the typical neuronal plasticity observed following CST transection was unaffected. Oligodendrocyte precursor cells (OPCs) were significantly increased at one week post injury in the IML of mice fed the control diet, with no change in mature oligodendrocytes (OLs). Following microglia elimination, the baseline population of OPCs in the IML was increased, suggesting increased OPC proliferation. Injury in the microglia depleted mice resulted in no additional increase in OPCs. Though baseline astrocyte activation and GFAP protein expression were unaffected, microglia elimination led to increased activation and GFAP protein post injury when compared with mice fed the control diet. These results reveal that microglia regulate the baseline OPC population in the uninjured spinal cord and that activated microglia influence the activities of OL lineage cells as well as astrocytes. The regulatory roles of microglia observed in this study likely contribute to the long term survival of the IML neurons observed following the distal axon injury.

Segregated neural explants exhibit co-oriented, asymmetric, neurite outgrowth.

Explants of embryonic chick sympathetic and sensory ganglia were found to exhibit asymmetric radial outgrowth of neurites under standard culture conditions with or without exogenous Nerve Growth Factor [NGF]. Opposing sides of an explant exhibited: a) differences in neurite length and, b) differences in neurite morphology. Strikingly, this asymmetry exhibited co-orientation among segregated, neighboring explants. The underlying mechanism(s) of the asymmetry and its co-orientation are not known but appear to depend on cell clustering because dissociated sympathetic neurons do not exhibit co-orientation whereas re-aggregated clusters of cells do. This emergent behavior may be similar to the community effect described in other cell types. If a similar phenomenon exists in the embryo, or in maturity, it may contribute to the establishment of proper orientation of neurite outgrowth during development and/or injury-induced neuronal plasticity.

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.

Retrograde influences of SCG axotomy on uninjured preganglionic neurons.

There is evidence that neuronal injury can affect uninjured neurons in the same neural circuit. The overall goal of this study was to understand the effects of peripheral nerve injury on uninjured neurons located in the central nervous system (CNS). As a model, we examined whether axotomy (transection of postganglionic axons) of the superior cervical ganglion (SCG) affected the uninjured, preganglionic neurons that innervate the SCG. At 7 days post-injury a reduction in choline acetyltransferase (ChAT) and synaptophysin immunoreactivity in the SCG, both markers for preganglionic axons, was observed, and this reduction persisted at 8 and 12 weeks post-injury. No changes were observed in the number or size of the parent cell bodies in the intermediolateral cell column (IML) of the spinal cord, yet synaptic input to the IML neurons was decreased at both 8 and 12 weeks post-injury. In order to understand the mechanisms underlying these changes, protein levels of brain-derived neurotrophic factor (BDNF) and tyrosine receptor kinase B (TrkB) were examined and reductions were observed at 7 days post-injury in both the SCG and spinal cord. Taken together these results suggest that axotomy of the SCG led to reduced BDNF in the SCG and spinal cord, which in turn influenced ChAT and synaptophysin expression in the SCG and also contributed to the altered synaptic input to the IML neurons. More generally these findings provide evidence that the effects of peripheral injury can cascade into the CNS and affect uninjured neurons.

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.

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.

Increased Cx32 expression in spinal cord TrkB oligodendrocytes following peripheral axon injury.

Following injury to motor axons in the periphery, retrograde influences from the injury site lead to glial cell plasticity in the vicinity of the injured neurons. Following the transection of peripherally located preganglionic axons of the cervical sympathetic trunk (CST), a population of oligodendrocyte (OL) lineage cells expressing full length TrkB, the cognate receptor for brain derived neurotrophic factor (BDNF), is significantly increased in number in the spinal cord. Such robust plasticity in OL lineage cells in the spinal cord following peripheral axon transection led to the hypothesis that the gap junction communication protein connexin 32 (Cx32), which is specific to OL lineage cells, was influenced by the injury. Following CST transection, Cx32 expression in the spinal cord intermediolateral cell column (IML), the location of the parent cell bodies, was significantly increased. The increased Cx32 expression was localized specifically to TrkB OLs in the IML, rather than other cell types in the OL cell lineage, with the population of Cx32/TrkB cells increased by 59%. Cx32 expression in association with OPCs was significantly decreased at one week following the injury. The results of this study provide evidence that peripheral axon injury can differentially affect the gap junction protein expression in OL lineage cells in the adult rat spinal cord. We conclude that the retrograde influences originating from the peripheral injury site elicit dramatic changes in the CNS expression of Cx32, which in turn may mediate the plasticity of OL lineage cells observed in the spinal cord following peripheral axon injury.

'Mature' nerve growth factor is a minor species in most peripheral tissues.

The classic neurotrophin hypothesis is based on the idea that innervating neurons derive 'mature' neurotrophin provided by the target for their survival. Yet large precursor forms of the neurotrophin nerve growth factor (NGF) have been reported in both central and peripheral tissues. In the present study, immunoblotting was used to survey peripheral tissues containing NGF-responsive neurons and to characterize various NGF species. These results demonstrate that 'mature' forms of NGF, i.e., the 13 and 16kDa species, are rare in sympathetic and sensory ganglia and in their peripheral targets, and that large molecular weight NGF precursors are abundant. In addition, certain NGF forms predominate in a given tissue, with each tissue exhibiting a characteristic NGF expression pattern. These findings suggest that NGF processing in peripheral tissues and in NGF-responsive ganglia may involve a variety of NGF species.

Reproductive activation of pine voles (Microtus pinetorum): examination of physiological markers.

We tested the hypothesis that the presence of an opposite-sex conspecific will result in time-related changes in measures of reproductive activation. We housed male-female pairs of pine voles together for 0, 2, 6, 12, or 24 h before collecting blood, reproductive organs and brains for immunocytochemical analysis of LHRH and c-fos. Control animals were never exposed to an opposite-sex conspecific. Following exposure to a male, there was a significant increase in uterine weight but not in LH levels. In males, there were no changes in peripheral indices of activation, i.e. LH levels, testes and seminal vesicle weights were not altered. Consistent with no change in circulating levels of LH, there was no change in LHRH immunoreactivity at any time. However, c-fos immunoreactivity was significantly greater in both males and females in the cingulate cortex and rostral bed nucleus of the stria terminalis (BNST) at 2 h, and in the caudal BNST at 2, 6 and 12 h. Similarly, c-fos immunoreactivity was increased in the rostral MPOA in both males and females at 2 and 6 h. However, in the caudal MPOA, there was a significant interaction between sex and time due to increased c-fos immunoreactivity in females only at 6 h. These results indicate that, in both male and female pine voles, exposure to an opposite-sex conspecific is sufficient to produce rapid, neural activation in brain areas known to be involved in reproductive activation and sexual behavior. This early activation did not occur in LHRH neurons. It is not known if this activation, particularly at early times, is due to reproductive activation or to the formation of pair bonds.

Distribution and phenotype of TrkB oligodendrocyte lineage cells in the adult rat spinal cord.

The distribution and phenotype of a previously undescribed population of nonneuronal cells in the intact spinal cord that expresses TrkB, the cognate receptor for brain derived neurotrophic factor (BDNF) and neurotrophin 4 (NT-4), were characterized by examining the extent of co-localization of TrkB with NG2, which identifies oligodendrocyte progenitors (OPCs) or CC1, a marker for mature oligodendrocytes (OLs). All TrkB nonneuronal cells expressed Olig2, confirming their role in the OL lineage. Similar to OPCs and OLs, TrkB cells resided in gray and white matter of the spinal cord in similar abundance. Less than 2% of TrkB cells expressed NG2, while over 80% of TrkB cells in the adult spinal cord co-expressed CC1. Most OPCs did not express detectable levels of TrkB, however a small OPC pool (~5%) showed TrkB immunoreactivity. The majority of mature OLs (~65%) expressed TrkB, but a population of mature OLs (~36%) did not express TrkB at detectable levels, and 17% of TrkB nonneuronal cells did not express NG2 or CC1. Approximately 20% of the TrkB nonneuronal population in the ventral horn resided in close proximity to motor neurons and were categorized as perineuronal. We conclude that TrkB is expressed by several pools of OL lineage cells in the adult spinal cord. These findings are important in understanding the neurotrophin regulation of OL lineage cells in the adult spinal cord.

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.

Transient changes in spinal cord glial cells following transection of preganglionic sympathetic axons.

Following peripheral nerve injury, retrograde signals originating from the injury site may activate intrinsic factors in the injured neurons, possibly leading to regenerative growth. Retrograde influences from peripheral injury sites may lead to the activation of glial cells in the vicinity of the centrally located cell bodies of the injured neurons. Few studies have examined changes in the spinal cord intermediolateral cell column (IML), which houses sympathetic preganglionic cell bodies, following injury to distal axons in the cervical sympathetic trunk (CST). The goal of the present study was to determine if transection of the CST results in plasticity in glial cells in the IML. At 1 day following injury, changes in the expression of microglial marker Iba1 were observed and the typical oligodendrocyte-neuronal relationship was altered. By 7 days, astrogliosis, microglial aggregation, and increased numbers of oligodendrocytes, as well as enhanced glial-glial and glial-neuronal relationships were present. The majority of cases were similar to controls at 3 weeks following injury and no changes were observed in any cases at 10 weeks following the injury. These results revealed changes in astrocytes, microglia, oligodendrocytes in the spinal cord following transection of preganglionic axons comprising the CST, indicating their ability to respond to distal axonal injury.

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.

Increased NGF proforms in aged sympathetic neurons and their targets.

Target-derived neurotrophins such as nerve growth factor (NGF) and neurotrophin-3 (NT-3) regulate sympathetic neuron survival. Here, NGF and NT-3 protein and transcript were examined in sympathetic neurons and targets in order to determine their role in age-related neuronal atrophy. One obvious alteration was a dramatic increase (up to 50-fold) in NGF protein forms, corresponding to proNGF-B, in the superior cervical ganglion (SCG) and targets where sympathetic innervation shows atrophy. In the iris, where sympathetic innervation is protected into old age, proNGF-B was decreased. Alterations in NGF transcript paralleled changes in NGF protein, albeit to a lesser degree. Though significantly increased in aged SCG, NT-3 protein, found primarily as the 'mature' form, showed only minor changes in most tissues, though NT-3 mRNA generally was decreased. In contrast, both NT-3 transcript and NT-3 precursors were increased in iris. The dramatic increases in proNGF, together with minimal changes in NT-3, suggest that alterations in NGF regulation may contribute to the loss of sympathetic innervation observed in many aged peripheral targets.