Francis J. Liuzzi

Long-Term Estrogen Replacement Coordinately Decreases trkA and β-PPT mRNA Levels in Dorsal Root Ganglion Neurons

Estrogen status has profound effects on cutaneous sensitivity in adult female rats. The presence of alpha-estrogen receptor mRNA and protein in NGF-dependent, adult female rat dorsal root ganglion (DRG) neurons raises the possibility that estrogen modulates cutaneous sensation by acting directly on primary afferent neurons, perhaps by altering their sensitivity to NGF. The present study examined the effect of long-term (90 days) daily injections of an estrogen preparation, Premarin (Wyeth-Ayerst, Radnor, PA), to ovariectomized adult rats on lumbar DRG high-affinity NGF receptor, trkA, mRNA levels, and on beta-preprotachykinin (beta-PPT) mRNA levels, which have been shown to be regulated by NGF. Two doses were used in the experiments, the higher dose being 10 times that of the lower dose. Such injections had an effect opposite that reported for short-term, acute estrogen treatment on DRG trkA mRNA levels. The current data show that long-term daily estrogen treatment decreases trkA mRNA levels by 36%. After 90 days of estrogen treatment, no dose effect was evident. Moreover, as would be expected if beta-PPT gene expression is regulated by NGF through the trkA receptor, long-term estrogen treatment decreased DRG neuronal beta-PPT mRNA levels by about 30%. As with trkA, there was no dose effect evident after 90 days of estrogen treatment. These data suggest the possibility that estrogen modulates DRG neuropeptide gene expression and, perhaps, cutaneous sensitivity by regulating NGF receptor gene expression.

Proteolysis is a critical step in the physiological stop pathway: mechanisms involved in the blockade of axonal regeneration by mammalian astrocytes.

Regenerating axons of adult dorsal roots are stopped by reactive astrocytes at the PNS-CNS junction. While it has been suggested that the astrocytes might pose a physical barrier to axonal growth, based on ultrastructural comparisons of physically blocked and axo-glial endings, it was proposed that astrocytes in the root transitional zone block axonal growth by activating the physiological stop pathway within the growing axon tips. Part of the stop pathway involves the proteolytic breakdown and removal of neurofilaments as they enter the axon endings. Another component involves the establishment of anterograde-to-retrograde conversion for the removal of membranous elements from the axonal endings. Both of these components appear to be dependent upon the activation of proteases within the axon tips. Therefore, to further test our hypothesis we infused, by intrathecal catheterization, the region of the dorsal root transitional zone with the protease inhibitor leupeptin at a time when the majority of regenerating axons have terminated in the region. Ultrastructural analyses after leupeptin treatment revealed axo-glial endings distended by accumulations of neurofilaments and organelles, particularly tubulovesicular profiles. These observations further support the idea that astrocytes, like normal target cells, can activate the physiological stop pathway.

Effects of short-term estrogen replacement on trkA mRNA levels in axotomized dorsal root ganglion neurons.

A population of adult dorsal root ganglion (DRG) neurons bind NGF with high affinity and express the trkA gene. In these cells, NGF regulates gene expression and function. Recently, a number of laboratories reported the presence of estrogen receptors in DRG neurons and profound effects of estrogen on DRG gene expression. Our laboratory, for example, has reported a significant and coordinate decrease in DRG trkA and beta-preprotachykinin (beta-PPT) mRNA levels following 90 days of daily estrogen injections to ovariectomized (OVX) rats. These data suggest, as has been suggested for medial septal cholinergic neurons, that estrogen may collaborate with NGF in the regulation of DRG neuronal gene expression and function. The current study examined further this potential collaboration in the DRG by determining the effect of short-term estrogen replacement in OVX rats on DRG trkA mRNA levels following sciatic nerve transection and the resulting removal of a vital source of NGF for those cells. In OVX rats, about 40% of lumbar DRG neurons contained trkA mRNA. Short-term estrogen replacement had no effect on the percentage of neurons containing trkA mRNA, but increased the mean trkA mRNA level in uninjured DRGs of OVX rats by 23%. Axotomy in OVX rats reduced the mean trkA mRNA level by 55% but did not significantly decrease the percentage of neurons containing the mRNA. Estrogen replacement, 7 days after axotomy, partially and significantly restored the mean trkA mRNA level. It was 49% greater than that of the untreated axotomized DRGs. It did not, however, significantly increase the percentage of DRG neurons containing trkA in axotomized DRGs. These observations show that short-term estrogen has an opposite effect on DRG neuronal trkA mRNA levels as compared to that of long-term estrogen demonstrated in our previous study. Moreover, the current data show that estrogen regulates trkA mRNA levels in the absence of target-derived NGF. These data suggest that estrogen may collaborate with NGF in the maintenance of normal adult DRG gene expression and function. Furthermore, these data suggest that loss of estrogen, such as that associated with menopause, may contribute to a decline in DRG neuronal function and an exacerbation of ongoing neuropathic processes.

Estrogen regulates neurofilament gene expression in adult female rat dorsal root ganglion neurons

Recently, adult female dorsal root ganglion (DRG) neurons were shown to express the estrogen receptor gene and to bind estrogen. This gene expression and binding is hormone dependent. Moreover, in a subpopulation of DRG neurons, the estrogen receptor is colocalized with high-affinity (trkA) and low-affinity (p75NGFR) receptors for nerve growth factor (NGF). In this NGF-responsive subpopulation of DRG neurons, estrogen regulates expression of the NGF receptor genes and may increase the sensitivity of these cells to the neurotrophin. The present study tested the hypothesis that neurofilament gene expression, which is regulated by NGF in these cells, is dependent on hormone status. In this study, ovariectomized (OVX) rats received either long-term physiological estrogen (conjugated estrogens; Premarin, Wyeth-Ayerst) replacement (low dose) or 10 times physiological levels (high dose). Quantitative in situ hybridization with an RNA probe for the 68-kDa neurofilament mRNA revealed a significant dose-dependent effect of Premarin on DRG neurofilament gene expression. In OVX animals receiving low-dose Premarin replacement therapy the mean steady-state 68-kDa mRNA level was as high as 4 times that of untreated OVX rats. High-dose therapy increased the mean 68-kDa neurofilament steady-state mRNA level to as much as six-fold that observed in untreated OVX animals. The estrogen-dependent upregulation of neurofilament gene expression appeared to occur in all DRG neurons, rather than in a subpopulation of those cells. These data suggest an important role for estrogen in the maintenance and function of primary sensory neurons. Whether estrogen directly regulates neurofilament gene expression or indirectly regulates it by increasing DRG neuronal sensitivity to neurotrophins or other growth factors remains to be determined.

Development of nitric oxide synthase expression in the superficial dorsal horn of the rat spinal cord

Development of nitric oxide synthase (NOS) expression in the superficial dorsal horn of the rat spinal cord was studied using NADPH diaphorase histochemistry. At birth, no positive staining was seen in the superficial laminae of the cord. A week later, a few small positive neurons and fibers were seen in presumptive lamina II. The adult pattern of NOS expression was evident by the end of the third postnatal week.

Differential Laminin Gene Expression in Dorsal Root Ganglion Neurons and Nonneuronal Cells

Laminin is a substrate for cell migration and process outgrowth during development and may play an important role in peripheral nerve regeneration. While most studies have emphasized the expression and production of this glycoprotein by Schwann cells, in the current study we use in situ hybridization to examine the expression of the various laminin chain genes in neuronal as well as nonneuronal cells of normal adult rat lumbar dorsal root ganglia. Hybridization using cRNA probes derived from cDNAs encoding nonhomologous regions of each laminin chain showed differential localization of the chain messages within the ganglia. B2 chain mRNA was abundant in large and small neurons, satellite cells, and Schwann cells. B1 chain message was also localized in satellite and Schwann cells, but its neuronal expression was primarily restricted to a subpopulation of smaller neurons. Message encoding merosin M chain, an A chain variant, was localized within satellite cells and Schwann cells but was absent or of very low abundance in neurons. A chain mRNA was not detectable in any of the three nervous tissue cell types. The differential expression of the four laminin chains in the DRG is discussed in relation to the functional epitopes of the molecule.

Radial glial interaction with cerebral germinal matrix capillaries in the fetal baboon

The occurrence of intraventricular hemorrhage (IVH) is a frequent problem in premature infants delivered between 24 (60%) and 32 weeks (80%) of gestation, a time during which the germinal matrix (GM) is prominent over the head of the caudate nucleus. Most IVH arises from the GM and it has been proposed that an important factor in the pathogenesis of IVH is a weakness of GM capillary walls due to deficient support by surrounding immature glial cells. The purpose of this study was to examine the glial-capillary interaction in the GM of fetal baboons sacrificed at 100 days (54%) gestation, a stage of GM development comparable to that during which human neonatal IVH occurs. Brains from a later gestational stage (162 days, 88%), after GM involution, were also examined. At 100 days of gestational age, the GM was prominent over the head of the caudate and contained vimentin positive, but not glial fibrillary acidic protein positive, radial glial cells which formed endfeet on capillaries in the region. Ultrastructurally, all the GM capillaries examined from this gestational time had complete, continuous endothelia marked by few pinocytotic vesicles and prominent tight junctions. The endothelial cells rested upon uninterrupted basement membranes which were contacted by clearly identifiable glial endfeet. These data show that GM capillaries have morphologies typical of CNS capillaries and suggest that the capillary immaturity within the GM is not a major contributing factor to IVH.

Streptozotocin-Induced Diabetes Mellitus Causes Changes in Primary Sensory Neuronal Cytoskeletal mRNA Levels That Mimic Those Caused by Axotomy☆

Dorsal root ganglion (DRG) sensory neurons are particularly vulnerable to diabetes mellitus. There is evidence that the disease decreases both circulating and retrogradely transported neurotrophic factors that are essential to the normal maintenance and function of these cells. A substantive loss of trophic support should cause DRG neurons to respond as though they were axotomized and, like an axotomy, cause significant changes in cytoskeletal gene expression within these cells. Such changes might contribute to the deficits in sensory neuronal function that characterize diabetic neuropathy. The current study used quantitative in situ hybridization to test the hypothesis that streptozotocin-induced diabetes, like an axotomy, increases class III beta-tubulin gene expression and decreases neurofilament 68-kDa gene expression in lumbar DRG neurons. In animals that had been diabetic for 8 weeks with mean blood glucose levels of 340 mg/dl, lumbar DRG class III beta-tubulin mRNA mean steady-state levels were twofold higher than those in age-matched nondiabetic controls. Moreover, in the same animals, diabetes decreased lumbar DRG 68-kDa neurofilament mRNA mean steady-state levels by more than half. These data show that diabetes causes changes in primary sensory neuronal cytoskeletal gene expression that mimic those caused by axotomy. Moreover, they support the idea that a loss of neurotrophic support contributes to the pathogenesis of diabetic neuropathy.

Up-regulation of laminin B2 gene expression in dorsal root ganglion neurons and nonneuronal cells during sciatic nerve regeneration.

A recent study in our laboratory showed that B2 laminin gene is expressed by all L4 and L5 dorsal root ganglion (DRG) neurons as well as by satellite and Schwann cells. Because the laminin B2 subunit has a domain that supports neurite extension in culture, the present study was undertaken to test the hypothesis that laminin B2 gene expression would increase during sciatic nerve regeneration. In situ hybridization was used to examine B2 laminin gene expression in L4 and L5 DRGs 28 days after creating and bridging a 10-mm sciatic nerve gap with an impermeable silicone tube. Overall there was a nearly threefold increase in DRG B2 laminin chain mRNA at this timepoint, a time when axons are known to show vigorous regrowth. Both neurons and nonneuronal cells contributed to this increase. These data suggest that an up-regulation of B2 laminin gene expression by DRG neurons and nonneuronal cells may play a role in peripheral nerve regeneration.

Short-Term Estrogen Replacement Increases β-Preprotachykinin mRNA Levels in Uninjured Dorsal Root Ganglion Neurons, but Not in Axotomized Neurons

Dorsal root ganglion (DRG) neurons that mediate nociception express the high affinity NGF receptor (trkA) gene and the preprotachykinin (PPT) gene. NGF has been shown to regulate both of these DRG neuronal genes. Our laboratory has shown that these genes are also regulated by estrogen. Long-term daily estrogen replacement, in adult ovariectomized (OVX) rats, causes a coordinate decline in trkA and beta-PPT mRNA levels in lumbar DRG neurons, while short-term estrogen replacement increases trkA mRNA levels in uninjured as well as in axotomized lumbar DRG neurons. The purpose of the current study was to test the hypothesis that short-term estrogen replacement increases DRG beta-PPT mRNA levels in lumbar DRG neurons of OVX rats and that the increase is dependent on target-derived NGF. Sciatic nerve transection (SNT) was used to eliminate target-derived NGF in L4 and L5 DRGs in adult OVX rats. Seven days later, one-half of the SNT and one-half of the animals that had received sham sciatic nerve transactions (SHAM) received two daily injections of estradiol benzoate (EB). The remaining rats received two daily injections of vehicle alone. Quantitative in situ hybridization analyses of sections from L4 and L5 DRGs showed that two daily injections of EB significantly increased beta-PPT mRNA levels in DRGs of SHAM animals, but had no effect on beta-PPT mRNA levels in DRGs from SNT animals. These data coupled with our earlier observations of the effect of short-term estrogen replacement on DRG trkA mRNA levels, indicate that the regulation of DRG beta-PPT mRNA levels by estrogen requires target-derived NGF.