Reduced Ia-afferent-mediated Hoffman reflex in streptozotocin-induced diabetic rats.

In addition to reduced nerve conduction velocity, diabetic neuropathic patients often exhibit a reduction in the amplitude of the compound muscle action potential elicited by stimulation of the Ia-afferent-mediated reflex pathway (Hoffman or H wave) that can contribute to diminished or absent tendon reflexes. In contrast to nerve conduction velocity deficits, changes in H-wave amplitudes have not been reproduced in diabetic animal models. Using electrophysiological techniques developed for repeated recordings in individual animals, we report H-wave deficits in streptozotocin (STZ)-treated insulin-dependent diabetic rats. After 4 weeks of diabetes induced by STZ treatment, a 47% reduction in the H-wave amplitude was demonstrated by recording compound muscle action potentials in foot muscles after stimulation of Ia afferents. Interestingly, we also demonstrate that the H-wave amplitude gradually recovers to a 26% deficit after 12 weeks of experimental diabetes. The recovery of the H wave in STZ-treated rats distinguishes this deficit mechanistically from other STZ-induced electrophysiological changes and may model a similar recovery of the H wave reported in diabetic patients.

The costimulatory molecule ICOS plays an important role in the immunopathogenesis of EAE.

The inducible costimulatory molecule (ICOS) is expressed on activated T cells and participates in a variety of important immunoregulatory functions. After the induction of experimental allergic encephalomyelitis in SJL mice with proteolipid protein (PLP), brain ICOS mRNA and protein were up-regulated on infiltrating CD3+ T cells before disease onset. ICOS blockade during the efferent immune response (9-20 days after immunization) abrogated disease, but blockade during antigen priming (1-10 days after immunization) exacerbated disease. Upon culture with PLP and compared with immunized controls, splenocytes produced either decreased interferon-gamma (IFN-gamma, in efferent blockade) or excessive IFN-gamma (in priming blockade). PLP-specific immunoglobulin G1 was decreased in animals treated with anti-ICOS during antigen priming, but not in other groups.

Comparison of the timing of acute blood-brain barrier breakdown to rabbit immunoglobulin G in the cerebellum and spinal cord of mice with experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is an animal model for human multiple sclerosis (MS). Similar to MS patients, EAE animals can exhibit chronic or relapsing, remitting paralysis; leukocyte infiltration of the central nervous system (CNS); and breakdown of the blood-brain barrier (BBB), allowing access to serum components. EAE pathology in rodents is generally thought to progress from the spinal cord to the more rostral brain. This common notion is based on numerous reports on the severity and progression of cellular infiltration and BBB breakdown during the course of disease. We studied opening of the BBB in EAE mice immunized to the proteolipid protein (PLP) peptide, PLP 139-151, with or without the use of pertussis toxin. Peripherally injected rabbit immunoglobulin G showed significant penetration through a compromised BBB in EAE mice and was observed throughout the parenchyma as well as intracellularly in multiple neuronal types. Results demonstrate the novel finding that the cerebellar BBB is dramatically and briefly comprised, even before breakdown of the BBB in the thoracolumbar spinal cord and prior to symptomatic disease. The demonstration of susceptibility in the cerebellum provides an important target for studying the factors predisposing certain CNS regions to autoimmune-related compromise of the BBB, such as MS.

Classical and novel directions in neurotrophin transport and research: anterograde transport of brain-derived neurotrophic factor by sensory neurons.

After the discovery of nerve growth factor, a classic model of neurotrophin action was developed. In this model, nerve endings compete for limited quantities of neurotrophic factors produced in neuronal target tissues. Neurotrophins are bound with high-affinity receptors expressed on the neuronal membrane and then endocytosed and retrogradely transported back to the cell body of responsive neurons. This classic model of target derived trophic support has been utilized to explain a wide range of trophic actions including effects on neuronal survival, terminal branching, and protein expression. However, a number of recent findings in the field of neurotrophin research cannot be explained using the classic model. In the peripheral nervous system (PNS), sensory neurons have been shown to contain mRNA for a member of the neurotrophin family, brain-derived neurotrophic factor (BDNF). Sensory neurons do not receive synaptic input so neurotrophin production by these cells does not fit into the classic target derived model. In contrast to target derived trophic support, BDNF produced by sensory neurons provides local autocrine and paracrine neurotrophic support in vitro. Furthermore, in vivo, sensory neurons transport BDNF in the anterograde direction away from the cell body, and opposite to the retrograde direction utilized in the classic model. Thus, out of necessity, a new direction for neurotrophin research has developed to study the production and anterograde transport of neurotrophins. The importance of this new mode of neurotrophin action in the PNS is indicated by results that implicate it in the response to pain, inflammation, and nerve injury.

Decreased accumulation of endogenous brain-derived neurotrophic factor against constricting sciatic nerve ligatures in streptozotocin-diabetic and galactose-fed rats.

Anterograde and retrograde trafficking of brain-derived neurotrophic factor (BDNF) was examined in streptozotocin-diabetic and galactose-fed rats by measuring accumulation of endogenous neurotrophin proximal and distal to two constricting sciatic nerve ligatures and by direct injection of radiolabeled neurotrophin into the sciatic nerve. Compared to controls, accumulation of endogenous BDNF proximal and distal to the ligatures as well as basal levels in non-ligated nerve segments were decreased in streptozotocin-diabetic and galactose-fed rats. Neither streptozotocin diabetes nor galactose intoxication affected the amount of 125I-labeled BDNF retrogradely transported to the DRG after injection into the sciatic nerve. These results suggest that reduced anterograde and retrograde accumulations of BDNF in experimental diabetes are not a result of impaired capacity for receptor-mediated transport.

Brain-derived neurotrophic factor improves blood glucose control and alleviates fasting hyperglycemia in C57BLKS-Lepr(db)/lepr(db) mice.

Systemic administration of brain-derived neurotrophic factor (BDNF) decreases nonfasted blood glucose in obese, non-insulin-dependent diabetic C57BLKS-Lepr(db)/lepr(db) (db/db) mice, with a concomitant decrease in body weight. By measuring percent HbA1c in BDNF-treated and pair-fed animals, we show that the effects of BDNF on nonfasted blood glucose levels are not caused by decreased food intake but reflect a significant improvement in blood glucose control. Furthermore, once established, this effect can persist for weeks after cessation of BDNF treatment. Oral glucose tolerance tests were performed to examine the effects of BDNF on blood glucose control in the fasted state and after an oral glucose challenge. BDNF treatment normalized fasting blood glucose from initially hyperglycemic levels and also showed evidence for beneficial, although less marked, effects on the ability to remove exogenous glucose from blood. One means to lower fasting blood glucose is to reduce the glucose output of peripheral tissues that normally play a part in the maintenance of fasting hyperglycemia. Because the liver is the major endogenous source of glucose in blood during fasting, and because hepatic weight and glucose output are increased in type 2 diabetes, we evaluated the effects of BDNF on liver tissue. BDNF reduced the hepatomegaly present in db/db mice, in association with reduced liver glycogen and reduced liver enzyme activity in serum, supporting the possible involvement of liver tissue in the mechanism of action for BDNF.

Neuronal injury increases retrograde axonal transport of the neurotrophins to spinal sensory neurons and motor neurons via multiple receptor mechanisms.

We investigated the retrograde axonal transport of 125I-labeled neurotrophins (NGF, BDNF, NT-3, and NT-4) from the sciatic nerve to dorsal root ganglion (DRG) sensory neurons and spinal motor neurons in normal rats or after neuronal injury. DRG neurons showed increased transport of all neurotrophins following crush injury to the sciatic nerve. This was maximal 1 day after sciatic nerve crush and returned to control levels after 7 days. 125I-BDNF transport from sciatic nerve was elevated with injection either proximal to the lesion or directly into the crush site and after transection of the dorsal roots. All neurotrophin transport was receptor-mediated and consistent with neurotrophin binding to the low-affinity neurotrophin receptor (LNR) or Trk receptors. However, transport of 125I-labeled wheat germ agglutinin also increased 1 day after sciatic nerve crush, showing that increased uptake and transport is a generalized response to injury in DRG sensory neurons. Spinal cord motor neurons also showed increased neurotrophin transport following sciatic nerve injury, although this was maximal after 3 days. The transport of 125I-NGF depended on the expression of LNR by injured motor neurons, as demonstrated by competition experiments with unlabeled neurotrophins. The absence of TrkA in normal motor neurons or after axotomy was confirmed by immunostaining and in situ hybridization. Thus, increased transport of neurotrophic factors after neuronal injury is due to multiple receptor-mediated mechanisms including general increases in axonal transport capacity.

Consistent repeated M- and H-Wave recording in the hind limb of rats.

Sensory and motor conduction velocities calculated from latencies of H reflexes and M waves in rat hind limbs have been used to assess experimental peripheral neuropathy. Amplitudes and latencies vary with recording location, and are seldom assessed directly. Using subcutaneous electrodes on the foot, we recorded consistent M waves and H reflexes while stimulating the sciatic or tibial nerve. The late wave disappeared when dorsal roots were cut, verifying that it was an H reflex. However, stimulus-response characteristics differed from those in humans: (a) the threshold was often higher than for M waves; (b) stimulus intensity eliciting a maximum H-reflex amplitude (Hmax) was often higher than adequate for a maximum M-wave amplitude; and (c) the amplitudes of H reflexes stimulated with intensities supramaximal for the M wave were over 90% of Hmax. H reflexes and M waves recorded repeatedly in rats can be useful in assessing sensory and motor function in models of neuropathy, using amplitudes as well as conduction velocities.

Axotomy upregulates the anterograde transport and expression of brain-derived neurotrophic factor by sensory neurons.

In addition to the known retrograde transport of neurotrophins, it is now evident that endogenous brain-derived neurotrophic factor (BDNF) is transported in the anterograde direction in peripheral and central neurons. We used a double-ligation procedure that distinguishes between anterograde and retrograde flow to quantify the anterograde transport of endogenous neurotrophins and neuropeptides in the peripheral nervous system before and after axotomy. BDNF accumulation proximal to the ligation (anterograde transport) was twice that distal to the ligation (retrograde direction). Anterograde transport of nerve growth factor and neurotrophin-3 was not evident. Furthermore, BDNF anterograde transport increased 3.5-fold within 24 hr after sciatic nerve injury or dorsal rhizotomy. Anterograde transport of substance P and calcitonin gene-related peptide decreased after peripheral nerve lesion, demonstrating that there was no generalized increase in anterograde transport. To determine the source of the anterogradely transported BDNF, we performed in situ hybridization in a variety of tissues before and after axotomy. Expression of BDNF mRNA in proximal nerve segments did not change with treatment, showing that the increased accumulation of BDNF was not a result of increased local synthesis. BDNF mRNA and protein were expressed by dorsal root ganglion sensory neurons but not by motor neurons. BDNF mRNA expression was increased 1 d after nerve injury, and BDNF protein was also increased twofold to threefold, suggesting that sensory neurons are the major contributing source of the increased BDNF traffic in the sciatic nerve. Our results suggest that increased anterogradely transported BDNF plays a role in the early neuronal response to peripheral nerve injury at sites distal to the cell body.

Effects of postnatal anti-NGF on the development of CGRP-IR neurons in the dorsal root ganglion.

Experiments were undertaken to examine anatomical correlates of physiological effects of rabbit sera raised against nerve growth factor (anti-NGF) on nociceptive afferents. This antiserum has been shown to deplete the population of A-delta high threshold mechanoreceptors and to reduce neurogenic vasodilatation. Because numerous studies implicate calcitonin gene related peptide (CGRP)-containing sensory neurons in these effects, immunocytochemical and anatomical techniques were used to examine the normal development of CGRP-immunoreactive (-IR) neurons in the dorsal root ganglion (DRG) of rats from 13 days to 19 weeks of age, and to compare this to the development in rats treated neonatally (postnatal days 2-14) with anti-NGF. In controls the rate of increase in the mean diameter of CGRP-IR cells was substantially greater between 13 days and 5 weeks of age than it was between 5 weeks and 19 weeks, in contrast to CGRP-negative neurons whose rate of growth remained relatively constant. Anti-NGF had no significant effect on growth rate, but rats treated with anti-NGF exhibited a reduced proportion of CGRP-IR neurons at 5 weeks. This deficit was reversed by 19 weeks unlike the physiological changes. These results indicate independent regulation of CGRP expression and nociceptor physiology by NGF.

Physiological characterization of Taxol-induced large-fiber sensory neuropathy in the rat.

The cancer chemotherapeutic agent Taxol (paclitaxel) causes a dose-related peripheral neuropathy in humans. We produced a dose-dependent large-fiber sensory neuropathy, without detrimental effects on general health, in mature rats by using two intravenous injections 3 days apart. Tests of other dosing schedules demonstrated the dependence of the severity of the neuropathy and of animal health on both the dose and the frequency of dosing. Pathologically, severe axonal degeneration and hypomyelination were observed in sections of dorsal roots, whereas ventral roots remained intact. Electrophysiologically, H-wave amplitudes in the hindlimb and amplitudes of predominantly sensory compound nerve action potentials in the tail were reduced. These effects persisted for at least 4 months after treatment. Motor amplitudes were not affected, but both motor and sensory conduction velocities decreased. The ability of rats to remain balanced on a narrow beam was impaired, indicating proprioceptive deficits. Muscle strength, measured by hindlimb and forelimb grip strength, and heat nociception, measured by tail-flick and hindlimb withdrawal tests, were not affected by Taxol. This model of Taxol-induced neuropathy in mature rats, with minimal effects on general health, parallels closely the clinical syndrome observed after Taxol treatment in humans.

Rabbit IgG distribution in skin, spinal cord and DRG following systemic injection in rat.

In order to determine the distribution of antibodies such as anti-NGF following systemic injection in neonates, immunocytochemical techniques were used to examine the localization of rabbit IgG in rat skin, DRG, and spinal cord after treatments with normal rabbit serum or purified rabbit IgG. Daily subcutaneous injections beginning on postnatal day 2 or on day 15 were given for three days. On the fourth day the animals were sacrificed and tissues were processed for rabbit IgG-IR. In the dorsal and ventral spinal cord, staining intensities suggest a substantial increase in the blood-brain barrier during the first two weeks after birth. Staining intensity in the epidermis of the glabrous skin from the hindpaw was substantially lower than in the adjacent dermis. In addition, IgG infrequently accumulated intracellularly in intensely stained patches in the epidermis. IgG was also able to reach relatively high intracellular concentrations in a small number of sensory neurons. The IgG staining pattern in the skin was similar when anti-NGF itself was administered to the animals. The results are discussed in the context of the effects of anti-NGF on the development of nociceptive afferents.