Pressure-induced pain: early sign of diabetes-associated impairment of insulin production in rats.

The lack of simple, non-invasive tests for a sub-clinical decline in insulin production hampers detection of early-stage type 1 pre-diabetes. Pressure pain withdrawal threshold (PPT) is a sensitive index of insulinopenia in diabetic and 'pre-diabetic' rats, but its ability to detect human insulin insufficiency is not known; if predictive, PPT testing of those at risk for diabetes would be warranted. To address this question, we used meta-analyses to demonstrate (i) a similar relationship between blood glucose and insulin levels in humans and diabetic rats and (ii) the predictive value of PPT for insulinopenia in a composite group (n=53) of control, streptozotocin (STZ)-diabetic (STZ-HG), and normoglycemic (STZ-NG) rats. The frequency distributions of pooled insulin levels (ng/ml) consisted of three sub-populations, with peak values of <0.5, 1.5+/-0.05, and 3.2+/-0.04. Using the 2.3rd percentile of the sub-population with the highest insulin level (2.81 ng/ml) as a cut-off to define insulinopenia, 40 animals (98% of STZ and 25% of controls) were identified with compromised insulin production. The frequency distribution of pooled PPT values also consisted of three sub-populations (peaks at 75.9+/-0.6 g, 97+/-0.3 g and 122+/-0.8 g), and when 106 g (the 2.3rd percentile of the most pressure-tolerant sub-population) was used as a cut-off, PPT measurements identified 92% of STZ-injected rats and 83% of rats with insulinopenia, as defined by 2.81 ng/ml insulin cut-off. Assuming similar between-species pain mechanisms, these findings support the potential usefulness of PPT measurements for detection of early-stage human type 1 diabetes.

The beta3 subunit of the Na+,K+-ATPase mediates variable nociceptive sensitivity in the formalin test.

It is widely appreciated that there is significant inter-individual variability in pain sensitivity, yet only a handful of contributing genetic variants have been identified. Computational genetic mapping and quantitative trait locus analysis suggested that variation within the gene coding for the beta3 subunit of the Na+,K+-ATPase pump (Atp1b3) contributes to inter-strain differences in the early phase formalin pain behavior. Significant strain differences in Atp1b3 gene expression, beta3 protein expression, and biophysical properties of the Na+,K+ pump in dorsal root ganglia neurons from resistant (A/J) and sensitive (C57BL/6J) mouse strains supported the genetic prediction. Furthermore, in vivo siRNA knockdown of the beta3 subunit produced strain-specific changes in the early phase pain response, completely rescuing the strain difference. These findings indicate that the beta3 subunit of the Na+,K+-ATPase is a novel determinant of nociceptive sensitivity and further supports the notion that pain variability genes can have very selective effects on individual pain modalities.

Pressure pain precedes development of type 2 disease in Zucker rat model of diabetes.

Decreased hind limb pressure pain threshold (PPT) is an early indicator of insulinopenia and neuropathy developing in STZ-rat models of type 1 diabetes and pre-diabetes. To test if pain on pressure is also a hallmark of compensated insulin resistance and type 2 diabetes in this work we measured PPT of Zucker lean (ZL), Zucker fatty (ZF) and Zucker fatty diabetic rats (ZDF; 8 animals per group). Using clinically accepted cut-off values for diagnosis of human diabetes and pre-diabetes, at 6th week of age (the study entry), all animals maintained random blood glucose within a normal range (< 7.9 mM). Over the following 4 weeks, the random glucose remained normal in lean and ZF rats; it however crossed 11 mM cut-off for the diagnosis of diabetes in all ZDF rats. With no detectable relation to blood glucose levels or changes throughout the study, lean, ZF and ZDF rats maintained respectively highest, intermediate and lowest PPT levels (83+/-1, 70+/-1 and 59+/-1 g; mean values for all tests per group). Thus in Zucker rat model, type 2 diabetes-associated impairment of nerve function precedes the development of hyperglycemia. Furthermore, since normoglycemic, but displaying decreased PPT, ZF rats were strongly hyperinsulinemic (plasma insulin concentration 30+/-4 ng/ml vs. 2.4+/-0.3 ng/ml in lean rats) these data suggest that hyperinsulinemia compensating for glucose metabolism might not restore compromised nerve function.

Early diabetic neuropathy: triggers and mechanisms.

Peripheral neuropathy, and specifically distal peripheral neuropathy (DPN), is one of the most frequent and troublesome complications of diabetes mellitus. It is the major reason for morbidity and mortality among diabetic patients. It is also frequently associated with debilitating pain. Unfortunately, our knowledge of the natural history and pathogenesis of this disease remains limited. For a long time hyperglycemia was viewed as a major, if not the sole factor, responsible for all symptomatic presentations of DPN. Multiple clinical observations and animal studies supported this view. The control of blood glucose as an obligatory step of therapy to delay or reverse DPN is no longer an arguable issue. However, while supporting evidence for the glycemic hypothesis has accumulated, multiple controversies accumulated as well. It is obvious now that DPN cannot be fully understood without considering factors besides hyperglycemia. Some symptoms of DPN may develop with little, if any, correlation with the glycemic status of a patient. It is also clear that identification of these putative non-glycemic mechanisms of DPN is of utmost importance for our understanding of failures with existing treatments and for the development of new approaches for diagnosis and therapy of DPN. In this work we will review the strengths and weaknesses of the glycemic hypothesis, focusing on clinical and animal data and on the pathogenesis of early stages and triggers of DPN other than hyperglycemia.

Phylogenetic preservation of alpha3 Na+,K+-ATPase distribution in vertebrate peripheral nervous systems.

The alpha(3) isoform of Na(+),K(+)-ATPase is uniquely expressed in afferent and efferent neurons innervating muscle spindles in the peripheral nervous system (PNS) of adult rats, but the distribution pattern of this isoform in other species has not been investigated. We compared expression of alpha(3) Na(+),K(+)-ATPase in lumbar dorsal root ganglia (DRG), spinal roots, and skeletal muscle samples of amphibian (frog), reptilian (turtle), avian (pigeon and chicken), and mammalian (mouse and human) species. In all species studied, the alpha(3) Na(+),K(+)-ATPase isoform was nonuniformly expressed in peripheral ganglia and nerves. In spinal ganglia, only 5-20% of neurons expressed this isoform, and, in avian and mammalian species, these alpha(3) Na(+),K(+)-ATPase-expressing neurons belonged to a subpopulation of large DRG neurons. In ventral root fibers of pigeons, mice, and humans, the alpha(3) Na(+),K(+)-ATPase was abundantly expressed predominantly in small myelinated axons. In skeletal muscle samples from turtles, pigeons, mice, and humans, alpha(3) Na(+),K(+)-ATPase was detected in intramuscular myelinated axons and in profiles of nerve terminals associated with the equatorial and polar regions of muscle spindle intrafusal fibers. These results show that the expression profiles for alpha(3) Na(+),K(+)-ATPase in the peripheral nervous system of a wide variety of vertebrate species are similar to the profile of rats and suggest that stretch receptor-associated expression of alpha(3) Na(+),K(+)-ATPase is preserved through vertebrate evolution.

Mechanical hyperalgesia correlates with insulin deficiency in normoglycemic streptozotocin-treated rats.

The triggers and pathogenesis of peripheral diabetic neuropathy are poorly understood, and this study evaluated the role of insulinopenia in nociceptive abnormalities in the streptozotocin (STZ) rat model of diabetes to test the hypothesis that, in addition to hyperglycemia, impairment of insulin signaling may be involved in progression of neuropathy. We measured blood glucose, plasma insulin, and sciatic nerve glucose and sorbitol levels, and withdrawal thresholds for hind limb pressure pain and heat pain in STZ-injected rats that developed hyperglycemia or remained normoglycemic. The pressure pain threshold did not change in vehicle-injected controls, but during the 2 weeks after STZ, it decreased by 25-40% in STZ-hyperglycemic and STZ-normoglycemic animals (P<0.05). Mean heat pain threshold did not change in STZ-normoglycemic rats, but increased by about 1.5 degrees C in STZ-hyperglycemic rats (P<0.05). These pain thresholds did not correlate with blood or nerve glucose or sorbitol levels, but both correlated with plasma insulin level in STZ-normoglycemic rats, and low-dose insulin replacement normalized the pressure threshold without affecting blood glucose level. Thus, at least one of early signs of diabetic neuropathy in STZ-treated rats, mechanical hyperalgesia, can be triggered by moderate insulinopenia, irrespective of glycemic status of the animals.

"Clock-scan" protocol for image analysis.

Comparative analysis of extra- and intracellular distributions of protein markers in immunohistochemical and immunofluorescent studies relies on techniques of image analysis. Line or region of interest pixel intensity scans are methods routinely used. However, although having good spatial resolution, linear pixel intensity scans fail to produce integral image of the cellular distribution of the label. On the other hand, the regions of interest scans have good integrative capacity but low spatial resolution. In this work, we describe a "clock-scan" protocol that, when applied to convex objects (such as neuronal cell bodies and the majority of cells in culture), combines advantages and circumnavigates limitations of the above-mentioned techniques. The protocol 1) collects multiple radial pixel intensity profiles scanned from the cell center to the periphery, 2) scales these profiles according to the cell radius measured in the direction of the scan, and finally, 3) averages these individual profiles into one integral radial pixel intensity profile. Because of scaling, the mean pixel intensity profiles produced by the clock-scan protocol depend on neither the cell size nor, within reasonable limits, the cell shape. This allows direct comparison or, if required, averaging or subtraction of profiles of different cells. We have successfully tested the clock-scan protocol in experiments with immunostained dorsal root ganglion neurons. In addition, the protocol seems to be equally applicable for studies in a variety of other preparations.

Target-determined expression of alpha3 isoform of the Na+,K+-ATPase in the somatic nervous system of rat.

Factors that determine the differential expression of isoforms of Na(+),K(+)-ATPase in the nervous system of vertebrates are not understood. To address this question we studied the expression of alpha(3) Na(+),K(+)-ATPase in the L5 dorsal root ganglia (DRG) of developing rat, the normal adult rat, and the adult rat after peripheral axotomy. During development, the first alpha(3) Na(+),K(+)-ATPase-positive DRG neurons appear by embryonic day 21. At birth, the L5 DRG have a full complement (14 +/- 2%) of these neurons. By 15 days after sciatic nerve transection in adult rat, the number of alpha(3) Na(+),K(+)-ATPase-positive DRG neurons and small myelinated L5 ventral root axons decreases to about 35% of control counts. These results combined with data from the literature suggest that the expression of alpha(3) Na(+),K(+)-ATPase by rat somatic neurons is determined by target-muscle spindle-derived factors.

Relevance of hyperglycemia to early mechanical hyperalgesia in streptozotocin-induced diabetes.

A modified von Frey filament test and an algesiometer paw pressure test were used to measure mechanical nociceptive withdrawal thresholds of the hind limb of control rats and rats injected with streptozotocin (STZ, 50 mg/kg). STZ treatment induced hyperglycemia (HG rats) in about 40% of treated animals. The rest of the STZ-treated and control rats remained normoglycemic (NG rats) throughout the entire experiment. No indications of mechanical hyperalgesia were observed in control groups of animals injected with physiological buffer only. However, both the behavioral tests used detected a 15-30% decrease in the mechanical nociceptive threshold of rats treated with STZ. Furthermore, mechanical nociceptive threshold changes were statistically indistinguishable between NG and HG rats. Glucose tolerance test did not reveal abnormalities of glucose metabolism in NG rats (compared to control animals). However, 1 week after STZ injection, the serum insulin level of NG rats was significantly lower than that of age-matched control rats (0.81 +/- 0.16 vs. 3.5 +/- 0.4 ng/mL; p < 0.01). These data strongly argue that systemic hyperglycemia is not the only factor triggering the development of mechanical hyperalgesia in the STZ rat model of diabetes. Other than hyperglycemia, consequences of insulinemia or insulinemia itself may play an important role in early impairment of mechanical nociception in this animal model.

Mechanical hyperalgesia in rat models of systemic and local hyperglycemia.

Mechanical hyperalgesia is an early symptom of diabetic neuropathy. To evaluate the mechanisms underlying this symptom, it was studied and compared in rat models of systemic and local hyperglycemia. Systemic hyperglycemia was induced by a single injection of streptozotocin (STZ, 50 mg/kg). Local hyperglycemia either in L(5) dorsal root ganglion (DRG) or a segment of the sciatic nerve at mid-thigh level was maintained by perfusion with 30-mM glucose solution delivered from a surgically implanted osmotic minipump. Mechanical hyperalgesia was assessed using modified von Frey filaments and hind limb withdrawal threshold measurements. During 2 weeks of STZ-induced diabetes rat systemic blood glucose level increased from 5.1+/-0.3 to 23+/-1.9 mM and limb withdrawal threshold decreased by approximately 30% bilaterally. During 2 weeks of local perfusion systemic blood glucose did not change; however, rats that underwent perfusion of the DRG or sciatic nerve with glucose exhibited a rapid (completed in approximately 1 week) 40-50% decrease in ipsilateral limb withdrawal threshold. Perfusion of the sciatic nerve with the normoglycemic buffer solution did not affect withdrawal thresholds. The aldose reductase inhibitor sorbinil (2.5 mg/ml) when added to 30-mM glucose perfusion solution prevented hyperalgesia. These data suggest that mechanical hyperalgesia in diabetic animals may, at least in part, result from focal injury caused by a direct toxic effect of glucose in the peripheral nervous system. These data also support the idea of activation of aldose reductase and polyol pathway as an important mechanism of hyperglycemia-induced impairment of nerve function.