Diabetes-induced microvascular complications at the level of the spinal cord: a contributing factor in diabetic neuropathic pain.

KEY POINTS: Diabetes is thought to induce neuropathic pain through activation of dorsal horn sensory neurons in the spinal cord. Here we explore the impact of hyperglycaemia on the blood supply supporting the spinal cord and chronic pain development. In streptozotocin-induced diabetic rats, neuropathic pain is accompanied by a decline in microvascular integrity in the dorsal horn. Hyperglycaemia-induced degeneration of the endothelium in the dorsal horn was associated with a loss in vascular endothelial growth factor (VEGF)-A165 b expression. VEGF-A165 b treatment prevented diabetic neuropathic pain and degeneration of the endothelium in the spinal cord. Using an endothelial-specific VEGFR2 knockout transgenic mouse model, the loss of endothelial VEGFR2 signalling led to a decline in vascular integrity in the dorsal horn and the development of hyperalgesia in VEGFR2 knockout mice. This highlights that vascular degeneration in the spinal cord could be a previously unidentified factor in the development of diabetic neuropathic pain. ABSTRACT: Abnormalities of neurovascular interactions within the CNS of diabetic patients is associated with the onset of many neurological disease states. However, to date, the link between the neurovascular network within the spinal cord and regulation of nociception has not been investigated despite neuropathic pain being common in diabetes. We hypothesised that hyperglycaemia-induced endothelial degeneration in the spinal cord, due to suppression of vascular endothelial growth factor (VEGF)-A/VEGFR2 signalling, induces diabetic neuropathic pain. Nociceptive pain behaviour was investigated in a chemically induced model of type 1 diabetes (streptozotocin induced, insulin supplemented; either vehicle or VEGF-A165 b treated) and an inducible endothelial knockdown of VEGFR2 (tamoxifen induced). Diabetic animals developed mechanical allodynia and heat hyperalgesia. This was associated with a reduction in the number of blood vessels and reduction in Evans blue extravasation in the lumbar spinal cord of diabetic animals versus age-matched controls. Endothelial markers occludin, CD31 and VE-cadherin were downregulated in the spinal cord of the diabetic group versus controls, and there was a concurrent reduction of VEGF-A165 b expression. In diabetic animals, VEGF-A165 b treatment (biweekly i.p., 20 ng g-1 ) restored normal Evans blue extravasation and prevented vascular degeneration, diabetes-induced central neuron activation and neuropathic pain. Inducible knockdown of VEGFR2 (tamoxifen treated Tie2CreERT2 -vegfr2flfl mice) led to a reduction in blood vessel network volume in the lumbar spinal cord and development of heat hyperalgesia. These findings indicate that hyperglycaemia leads to a reduction in the VEGF-A/VEGFR2 signalling cascade, resulting in endothelial dysfunction in the spinal cord, which could be an undiscovered contributing factor to diabetic neuropathic pain.

Regulation of alternative VEGF-A mRNA splicing is a therapeutic target for analgesia.

Vascular endothelial growth factor-A (VEGF-A) is best known as a key regulator of the formation of new blood vessels. Neutralization of VEGF-A with anti-VEGF therapy e.g. bevacizumab, can be painful, and this is hypothesized to result from a loss of VEGF-A-mediated neuroprotection. The multiple vegf-a gene products consist of two alternatively spliced families, typified by VEGF-A165a and VEGF-A165b (both contain 165 amino acids), both of which are neuroprotective. Under pathological conditions, such as in inflammation and cancer, the pro-angiogenic VEGF-A165a is upregulated and predominates over the VEGF-A165b isoform. We show here that in rats and mice VEGF-A165a and VEGF-A165b have opposing effects on pain, and that blocking the proximal splicing event - leading to the preferential expression of VEGF-A165b over VEGF165a - prevents pain in vivo. VEGF-A165a sensitizes peripheral nociceptive neurons through actions on VEGFR2 and a TRPV1-dependent mechanism, thus enhancing nociceptive signaling. VEGF-A165b blocks the effect of VEGF-A165a. After nerve injury, the endogenous balance of VEGF-A isoforms switches to greater expression of VEGF-Axxxa compared to VEGF-Axxxb, through an SRPK1-dependent pre-mRNA splicing mechanism. Pharmacological inhibition of SRPK1 after traumatic nerve injury selectively reduced VEGF-Axxxa expression and reversed associated neuropathic pain. Exogenous VEGF-A165b also ameliorated neuropathic pain. We conclude that the relative levels of alternatively spliced VEGF-A isoforms are critical for pain modulation under both normal conditions and in sensory neuropathy. Altering VEGF-Axxxa/VEGF-Axxxb balance by targeting alternative RNA splicing may be a new analgesic strategy.

Ovarian VEGF(165)b expression regulates follicular development, corpus luteum function and fertility.

Angiogenesis and vascular regression are critical for the female ovulatory cycle. They enable progression and regression of follicular development, and corpora lutea formation and regression. Angiogenesis in the ovary occurs under the control of the vascular endothelial growth factor-A (VEGFA) family of proteins, which are generated as both pro-(VEGF(165)) and anti(VEGF(165)b)-angiogenic isoforms by alternative splicing. To determine the role of the VEGF(165)b isoforms in the ovulatory cycle, we measured VEGF(165)b expression in marmoset ovaries by immunohistochemistry and ELISA, and used transgenic mice over-expressing VEGF(165)b in the ovary. VEGF(165)b was expressed in the marmoset ovaries in granulosa cells and theca, and the balance of VEGF(165)b:VEGF(165) was regulated during luteogenesis. Mice over-expressing VEGF(165)b in the ovary were less fertile than wild-type littermates, had reduced secondary and tertiary follicles after mating, increased atretic follicles, fewer corpora lutea and generated fewer embryos in the oviduct after mating, and these were more likely not to retain the corona radiata. These results indicate that the balance of VEGFA isoforms controls follicle progression and luteogenesis, and that control of isoform expression may regulate fertility in mammals, including in primates.

Effects of acute abstinence and nicotine administration on taste perception in cigarette smokers.

We investigated the effects of short-term abstinence from smoking and acute nicotine administration on taste perception in smokers. We assessed sensitivity for salt and sucrose solutions and the self-reported intensity and pleasantness of these tastes, using a previously validated model of taste perception. This was in order to investigate mechanisms by which cigarette smoking and smoking cessation may modulate dietary behaviour. Male and female daily smokers attended a single testing session. Participants were randomised to either abstain for smoking for 12 h or smoke as usual on the morning of testing. At the testing session, participants completed subjective ratings of mood and ratings of intensity and pleasantness of salt and sucrose solutions, followed by measurement of the threshold at which these solutions could be detected on the tongue. Participants were then randomised to smoking either a nicotine-containing or denicotinised cigarette, after which they completed the same measures as previously. Our data suggest that following cigarette smoking, lower taste thresholds are obtained after smoking a denicotinised cigarette compared with a nicotinised cigarette, but among females only. This effect was not observed among males and did not differ as a function of abstinence condition. In addition, among non-abstinent smokers, females demonstrated higher taste thresholds (i.e. reduced sensitivity) for salt than males, but this sex difference was not observed among abstinent smokers.

Specific testicular cellular localization and hormonal regulation of the PKIalpha and PKIbeta isoforms of the inhibitor protein of the cAMP-dependent protein kinase.

We have previously demonstrated that there exist two distinct genes for the thermostable inhibitor protein of the cAMP-dependent protein kinase, PKIalpha and PKIbeta (Van Patten, S. M., Howard, P., Walsh, D. A., and Maurer, R. A. (1992) Mol. Endocrinol. 6, 2114-2122). We have also shown that in the testis, at least eight forms of PKIbeta exist, differing as a result of at least post-translational modification and alternate translational initiation (Kumar, P., Van Patten, S. M., and Walsh, D. A. (1997) J. Biol. Chem. 272, 20011-20020). We now report that in the testis, there is a unique cellular distribution of protein kinase inhibitor forms, with PKIbeta being essentially (if not exclusively) a germ cell protein and PKIalpha being expressed primarily in Sertoli cells. Furthermore, there is a progressive change in the forms of PKIbeta that are present within germ cells with development that is initiated in testis tubules and continues as the germ cells migrate through the epididymis. These conclusions are derived from studies with isolated cell populations and with the at/at germ cell-deficient mouse line, by in situ hybridization, and by following the developmental expression of these proteins in both testis and epididymis. We have also shown that follicle-stimulating hormone (FSH) can increase the expression of both PKIalpha and PKIbeta. The FSH-regulated expression of PKIalpha in the Sertoli cell likely occurs via the normal route of second messenger signal transduction. In contrast, the FSH-dependent PKIbeta expression must arise by some form of Sertoli cell-germ cell intercommunication.

Molecular characterization of a putative K-Cl cotransporter in rat brain. A neuronal-specific isoform.

Using a combination of data base searching, polymerase chain reaction, and library screening, we have identified a putative K-Cl cotransporter isoform (KCC2) in rat brain that is specifically localized in neurons. A cDNA of 5566 bases was obtained from overlapping clones and encoded a protein of 1116 amino acids with a deduced molecular mass of 123.6 kDa. Over its full length, the amino acid sequence of KCC2 is 67% identical to the widely distributed K-Cl cotransporter isoform (KCC1) identified in rat brain and rabbit kidney (Gillen, C., Brill, S., Payne, J.A., and Forbush, B., III(1996) J. Biol. Chem. 271, 16237-16244) but only approximately25% identical to other members of the cation-chloride cotransporter gene family, including "loop" diuretic-sensitive Na-K-Cl cotransport and thiazide-sensitive Na-Cl cotransport. Based on analysis of the primary structure as well as homology with other cation-chloride cotransporters, we predict 12 transmembrane segments bounded by N- and C-terminal cytoplasmic regions. Four sites for N-linked glycosylation are predicted on an extracellular intermembrane loop between putative transmembrane segments 5 and 6. Northern blot analysis using a KCC2-specific cDNA probe revealed a very highly expressed approximately5.6-kilobase transcript only in brain. Reverse transcriptase-polymerase chain reaction revealed that KCC1 was present in rat primary astrocytes and rat C6 glioma cells but that KCC2 was completely absent from these cells, suggesting KCC2 was not of glial cell origin. In situ hybridization studies demonstrated that the KCC2 transcript was expressed at high levels in neurons throughout the central nervous system, including CA1-CA4 pyramidal neurons of the hippocampus, granular cells and Purkinje neurons of the cerebellum, and many groups of neurons throughout the brainstem.

Neuropeptide gene expression and capsaicin-sensitive primary afferents: maintenance and spread of adjuvant arthritis in the rat.

1. Many experimental and clinical arthritides are characterized by their bilateral nature. There is strong evidence to suggest that this bilateral spread may be mediated by a neuronal mechanism. We have previously shown early and sustained induction of mRNAs encoding preprotachykinin (PPT) and calcitonin gene-related peptide (CGRP) in dorsal root ganglion (DRG) neurons innervating an inflamed, arthritic joint. We have now investigated the involvement of capsaicin-sensitive primary afferents and the expression of neuropeptide mRNAs in the maintenance and bilateral spread of mild adjuvant-induced arthritis in the rat. 2. Capsaicin was applied perineurally to either the left (Cap-L) or right (Cap-R) sciatic nerve of halothane-anaesthetized male Han Wistar rats. Two weeks after capsaicin lesioning, arthritis was induced by injection of Freund's complete adjuvant (FCA) around the left ankle at a dose that caused inflammation of the left ankle joint, and a delayed (14 days) contralateral (right) ankle arthritis. Arthritis was monitored for 15 days after injection, when animals were killed and the lumbar DRG dissected. PPT, CGRP, somatostatin (SS), and vasoactive intestinal polypeptide (VIP) mRNA expression was determined in L5 DRG using in situ hybridization. 3. Spread of inflammation/arthritis to the right limb was associated with bilateral rises in PPT and CGRP mRNA expression in L5 DRG. SS mRNA expression in right DRG was unaffected by spread of inflammation. FCA-L+Cap-L reduced left joint swelling and prevented spread of arthritis to the right joint when assessed by joint swelling. This inhibition of spread of arthritis was associated with significant reductions in all left L5 DRG neuropeptide mRNAs compared with controls, and the rise in right L5 DRG PPT mRNA expression seen in FCA-L-alone animals was blocked. FCA-L+Cap-R also reduced left joint swelling and prevented the spread of inflammation to the right ankle. This lesion prevented the rise in PPT and CGRP mRNA expression seen in right DRG with FCA-L alone. 4. These findings suggest a role for capsaicin-sensitive primary afferents and the primary afferent neuropeptides encoded by PPT and CGRP mRNA in the maintenance and spread of arthritis.

Increased expression of preprotachykinin, calcitonin gene-related peptide, but not vasoactive intestinal peptide messenger RNA in dorsal root ganglia during the development of adjuvant monoarthritis in the rat.

Neuropeptides in dorsal root ganglia (DRG) have been implicated in the pathogenesis of pain and neurogenic inflammation in experimental and clinical arthritis. Recently we demonstrated increased levels of substance P (SP) and calcitonin gene-related peptide (CGRP) confined to innervating DRG in adjuvant-mediated monoarthritis. We have now investigated whether changes in peptide content are reflected in altered neuropeptide gene expression and the time course involved. Using in situ hybridization we found marked increases in expression of beta-preprotachykinin (PPT; 81 +/- 24% rise) and alpha-CGRP (44 +/- 6% rise) mRNAs in innervating (ipsilateral L5) DRG neurones only. These increases occurred at the onset of acute inflammation (8 h) and persisted until chronic arthritis developed after 14 days. There were no changes in the proportion of DRG neurones expressing PPT or CGRP mRNAs. Messenger RNA encoding vasoactive intestinal polypeptide (VIP) was not induced. These data suggest that increased synthesis of PPT and CGRP peptides in DRG may play a role in the pathogenesis both of adjuvant-mediated acute inflammation and chronic arthritis.