Treatment of cryptoglandular fistulas with the fistula tract laser closure (FiLaC™) method in comparison with standard methods: first results of a multicenter retrospective comparative study in the Netherlands.

BACKGROUND: Current surgical closure techniques for sphincter-sparing treatment of high cryptoglandular fistulas in the Netherlands include the mucosal advancement flap procedure (MAF) and ligation of the intersphincteric fistula tract (LIFT). A relatively novel treatment is the fistula tract laser closure (FiLaC™) method. The aim of this study was to investigate the differences in healing and recurrence rates between FiLaC™ and current standard practices. METHODS: This multicenter retrospective cohort study included both primary and recurrent high cryptoglandular anorectal fistulas, treated with either FiLaC™ or standard methods (MAF or LIFT) between September 2015 and July 2020. Patients with extrasphincteric fistulas, Crohn's disease, multiple fistulas, age < 18 years or missing data regarding healing time or recurrence were excluded. The primary outcomes were the clinical primary and secondary healing and recurrence rates. Primary healing was defined as a closed external opening without fluid discharge within 6 months of treatment on examination, while secondary healing was the same endpoint after secondary treatment. Secondary outcomes included healing time and complaints. RESULTS: A total of 162 high fistulas from 3 Dutch hospitals were included. Ninety-nine high fistulas were treated with FiLaC™ and 63 with either MAF or LIFT. There were no significant differences between FiLaC™ and MAF/LIFT in terms of clinical healing (55.6% versus 58.7%, p = .601), secondary healing (70.0% versus 69.2%, p = .950) or recurrence rates (49.5% versus 54%, p = .420), respectively. Median follow-up duration was 7.1 months in the FiLaC™ group (interquartile range [IQR] 4.1-14.4 months) versus 6 months in the control group (IQR 3.5-8.1 months). CONCLUSIONS: FiLaC™ treatment of high anorectal fistulas does not appear to be inferior to MAF or LIFT. Based on these preliminary results, FiLaC™ can be considered as a worthwhile treatment option for high cryptoglandular fistulas. Prospective studies with a longer follow-up period and well-determined postoperative parameters such as complication rates, magnetic resonance imaging for confirmation of fistula healing, incontinence and quality of life are warranted.

Nerve growth factor-induced changes in the intracellular localization of the protein kinase C substrate B-50 in pheochromocytoma PC12 cells.

High levels of the neuron-specific protein kinase C substrate, B-50 (= GAP43), are present in neurites and growth cones during neuronal development and regeneration. This suggests a hitherto nonelucidated role of this protein in neurite outgrowth. Comparable high levels of B-50 arise in the pheochromocytoma PC12 cell line during neurite formation. To get insight in the putative growth-associated function of B-50, we compared its ultrastructural localization in naive PC12 cells with its distribution in nerve growth factor (NGF)- or dibutyryl cyclic AMP (dbcAMP)-treated PC12 cells. B-50 immunogold labeling of cryosections of untreated PC12 cells is mainly associated with lysosomal structures, including multivesicular bodies, secondary lysosomes, and Golgi apparatus. The plasma membrane is virtually devoid of label. However, after 48-h NGF treatment of the cells, B-50 immunoreactivity is most pronounced on the plasma membrane. Highest B-50 immunoreactivity is observed on plasma membranes surrounding sprouting microvilli, lamellipodia, and filopodia. Outgrowing neurites are scattered with B-50 labeling, which is partially associated with chromaffin granules. In NGF-differentiated PC12 cells, B-50 immunoreactivity is, as in untreated cells, also associated with organelles of the lysosomal family and Golgi stacks. B-50 distribution in dbcAMP-differentiated cells closely resembles that in NGF-treated cells. The altered distribution of B-50 immunoreactivity induced by differentiating agents indicates a shift of the B-50 protein towards the plasma membrane. This translocation accompanies the acquisition of neuronal features of PC12 cells and points to a neurite growth-associated role for B-50, performed at the plasma membrane at the site of protrusion.

Phosphorylation of the presynaptic protein B-50 (GAP-43) is increased during electrically induced long-term potentiation.

The protein B-50 (F1, GAP-43) is a presynaptic-specific substrate of protein kinase C, functionally related to neurotransmitter release. An increase in phosphorylation of this protein has been proposed as a molecular mechanism underlying long-term potentiation (LTP). B-50 phosphorylation measured by quantitative immunoprecipitation in rat hippocampal slices incubated in the presence of radiolabeled inorganic phosphate was increased for at least 1 hr after the induction of LTP in the CA1 region. No significant changes in B-50 phosphorylation were observed in untetanized slices stimulated at low frequency. The direct demonstration of an increased phosphorylation of the protein B-50 during LTP is consistent with the hypothesis that presynaptic mechanisms contribute to maintenance of LTP.

DOC2 proteins in rat brain: complementary distribution and proposed function as vesicular adapter proteins in early stages of secretion.

DOC2 proteins constitute a novel protein family that may function in secretion and contain a double C2 domain. We have cloned and characterized two DOC2 isoforms in rat brain and studied their interactions with other proteins implicated in secretion. DOC2A was virtually brain specific, DOC2B ubiquitous. Within brain, the isoforms were expressed nonuniformly and complementary within neurons, not astroglia, and copurified with synaptic vesicles. Affinity purification, yeast two-hybrid analysis, and coimmunoprecipitation revealed that DOC2 binds munc18, a protein also implicated in secretion. The first DOC2 C2 domain and most of munc18 are involved in direct interactions. Munc18 may regulate formation of 'core complexes' during vesicle docking, by interacting with syntaxin. We show that DOC2 and syntaxin compete for munc18. Other core complex components shifted the equilibrium between syntaxin-munc18 versus DOC2-munc18. These data suggest that DOC2 proteins are vesicular adapter proteins regulating munc18-syntaxin complexes and herewith synaptic vesicle docking.

Peripheral and central neurologic complications in type 2 diabetes mellitus: no association in individual patients.

Diabetes mellitus is associated with end-organ complications in the peripheral and central nervous system. It is unknown if these complications share a common aetiology, and if they co-occur in the same patient. The aim of the present study was to relate different measures of peripheral neuropathy in patients with type 2 diabetes mellitus (DM2) to cognition and brain MRI. A standardized neurological examination and questionnaire, neuropsychological examination and brain MRI were performed in 122 patients with DM2 and 56 matched controls. Measures of peripheral neuropathy were vibration threshold, a sensory examination sum score and the Toronto Clinical Neuropathy Scoring System. Neuropsychological test scores were expressed in standardized z-values across five predetermined cognitive domains. White matter lesions and cortical and subcortical atrophy were rated on MRI. Overall 38% of the patients with DM2 and 12% of the controls were classified as having any neuropathy (p<0.001). Patients with DM2 had a lower performance on the neuropsychological tests, more white matter lesions (p<0.01) and more atrophy (p<0.01) than controls. Within the DM2 group none of the measures of peripheral neuropathy was related to MRI abnormalities or cognitive dysfunction (linear regression analyses, adjusted for age, education, sex). We conclude that peripheral neuropathy in patients with DM2 is not related to cognitive dysfunction and brain abnormalities. This indicates that central and peripheral neurological complications of DM2 might have different etiologies.

B-50/GAP-43-induced formation of filopodia depends on Rho-GTPase.

In the present study we show that expression of the neural PKC-substrate B-50 (growth-associated protein [GAP-43]) in Rat-1 fibroblasts induced the formation of filopodial extensions during spreading. This morphological change was accompanied by an enhanced formation of peripheral actin filaments and by accumulation of vinculin immunoreactivity in filopodial focal adhesions, colocalizing with B-50. In time lapse experiments, the B-50-induced filopodial extensions were shown to stay in close contact with the substratum and appeared remarkably stable, resulting in a delayed lamellar spreading of the fibroblasts. The morphogenetic effects of the B-50 protein were entirely dependent on the integrity of the two N-terminal cysteines involved in membrane association (C3C4), but were not significantly affected by mutations of the PKC-phosphorylation site (S41) or deletion of the C terminus (177-226). Cotransfection of B-50 with dominant negative Cdc42 or Rac did not prevent B-50-induced formation of filopodial cells, whereas this process could be completely blocked by cotransfection with dominant negative Rho or Clostridium botulinum C3-transferase. Conversely, constitutively active Rho induced a similar filopodial phenotype as B-50. We therefore propose that the induction of surface extensions by B-50 in spreading Rat-1 fibroblasts depends on Rho-guanosine triphosphatase function.

Cognition and synaptic plasticity in diabetes mellitus.

Diabetes mellitus is associated with cognitive deficits and an increased risk of dementia, particularly in the elderly. These deficits are paralleled by neurophysiological and structural changes in the brain. In animal models of diabetes, impairments of spatial learning occur in association with distinct changes in hippocampal synaptic plasticity. At the molecular level these impairments might involve changes in glutamate-receptor subtypes, in second-messenger systems and in protein kinases. The multifactorial pathogenesis of diabetic encephalopathy is not yet completely understood, but clearly shares features with brain ageing and the pathogenesis of diabetic neuropathy. It involves both metabolic and vascular changes, related to chronic hyperglycaemia, but probably also defects in insulin action in the brain. Treatment with insulin might therefore not only correct hyperglycaemia, but could also directly affect the brain.

B-50, the growth associated protein-43: modulation of cell morphology and communication in the nervous system.

The growth-associated protein B-50 (GAP-43) is a presynaptic protein. Its expression is largely restricted to the nervous system. B-50 is frequently used as a marker for sprouting, because it is located in growth cones, maximally expressed during nervous system development and re-induced in injured and regenerating neural tissues. The B-50 gene is highly conserved during evolution. The B-50 gene contains two promoters and three exons which specify functional domains of the protein. The first exon encoding the 1-10 sequence, harbors the palmitoylation site for attachment to the axolemma and the minimal domain for interaction with G0 protein. The second exon contains the "GAP module", including the calmodulin binding and the protein kinase C phosphorylation domain which is shared by the family of IQ proteins. Downstream sequences of the second and non-coding sequences in the third exon encode species variability. The third exon also contains a conserved domain for phosphorylation by casein kinase II. Functional interference experiments using antisense oligonucleotides or antibodies, have shown inhibition of neurite outgrowth and neurotransmitter release. Overexpression of B-50 in cells or transgenic mice results in excessive sprouting. The various interactions, specified by the structural domains, are thought to underlie the role of B-50 in synaptic plasticity, participating in membrane extension during neuritogenesis, in neurotransmitter release and long-term potentiation. Apparently, B-50 null-mutant mice do not display gross phenotypic changes of the nervous system, although the B-50 deletion affects neuronal pathfinding and reduces postnatal survival. The experimental evidence suggests that neuronal morphology and communication are critically modulated by, but not absolutely dependent on, (enhanced) B-50 presence.

Reduced glutathione protects against cisplatin-induced neurotoxicity in rats.

Reduced glutathione (GSH) is reported to diminish cisplatin-induced neurotoxicity, and it was for this reason that we studied GSH in an animal model of cisplatin neuropathy. The neuropathy was evaluated by measuring the sensory nerve conduction velocity (SNCV) in young adult Wistar rats. GSH injections (i.v.) were given twice weekly, within five minutes before cisplatin was injected (i.p.). In a first experiment GSH (500 mg/kg) in combination with a low-dose cisplatin treatment (1 mg/kg, 10 weeks) was investigated. Animals treated with cisplatin and placebo developed a neuropathy (SNCV at week 10: age controls, 61.9 m/s; cisplatin alone, 44.2 m/s), whereas rats treated with cisplatin and GSH did not (SNCV, 61.9 m/s). The same dose of GSH was used in combination with a high-dose cisplatin schedule (2 mg/kg, 5 weeks' treatment plus 5 weeks' recovery). Again, GSH protected animals against the development of neuropathy (SNCV at week 10: age controls, 61.9 m/s; cisplatin alone, 50.6 m/s; cisplatin plus GSH, 61.1 m/s). In another experiment four lower doses of GSH (25, 50, 100, and 200 mg/kg) were tested in combination with the low-dose cisplatin protocol (1 mg/kg, 11 weeks). The cisplatin group developed a neuropathy (SNCV at week 11: cisplatin alone, 50.2 m/s; age controls, 60.6 m/s). Only the dose of 200 mg GSH/kg was found to protect against the development of a neuropathy (SNCV, 61.0 m/s). In an antitumor study GSH administered at 300 mg/kg in combination with cisplatin at 1.5 mg/kg did not diminish the curative effect of cisplatin. We conclude that GSH prevents cisplatin-induced neuropathy and that it should be investigated further in the clinic.

Morphological and electrophysiological study of the effects of cisplatin and ORG.2766 on rat spinal ganglion neurons.

Eleven- to 12-wk-old rats were treated twice a week with cisplatin/saline or with cisplatin plus ORG.2766 during 12.5 wk. Cisplatin and ORG.2766 were administered at a final concentration of 0.04 mg/ml (i.p.) and 10 micrograms/ml (s.c.), respectively. Control animals were treated with saline. In this period the cisplatin-treated animals developed a peripheral neuropathy resulting in impairment of sensory functions. Estimates of the motor (MNCV) and sensory (SNCV) nerve conduction velocity were made after 0, 7.5, 10, and 12.5 wk. It appeared that the MNCV of the control, cisplatin-, and cisplatin plus ORG.2766-treated rats increased from 50 to 59 m/s. In contrast, the SNCV of the cisplatin-treated rats decreased significantly (P less than 0.001) from 63 to 56 m/s, whereas that of the control animals increased from 62 to 84 m/s. Rats which received cisplatin plus ORG.2766 showed an increase in SNCV up to control levels. After 12.5 wk the animals were perfused with a mixture of 1% paraformaldehyde and 1.25% glutaraldehyde in 0.05 M phosphate buffer. At the level of L5 and L6, 5 mm of spinal cord tissue and three dorsal root ganglia were removed and processed for electron microscopy. With the point-counting method the volume fraction (v/v) of somata and myelin in spinal ganglia was estimated. No significant change in the volume fraction of somata of the control (0.42), cisplatin (0.33)-, and cisplatin plus ORG.2766 (0.39)-treated rats was found. The same held true for the volume fraction of myelin of the control (0.53), cisplatin (0.59)-, and cisplatin plus ORG.2766 (0.58)-treated rats. In addition, the number of lysosomes per 100 microns 2 was estimated in spinal ganglion neurons and in spinal cord motor neurons of a total of 120 randomly chosen neurons. It was found that the number of lysosomes in the spinal ganglion neurons of the control animals was lower (10 per 100 microns 2) than in cisplatin-treated (30 per 100 microns 2) and in cisplatin plus ORG.2766-treated rats (28 per 100 microns 2) (P less than 0.05). No difference was observed in the number of lysosomes between cisplatin- and cisplatin plus ORG.2766-treated rats. The number of lysosomes in spinal cord tissue of cisplatin-treated rats (2.4 per 100 microns 2) did not differ from controls (0.1 per 100 microns 2) and from cisplatin plus ORG.2766-treated rats (0.8 per 100 microns 2).(ABSTRACT TRUNCATED AT 400 WORDS)

Antagonism of the melanocortin system reduces cold and mechanical allodynia in mononeuropathic rats.

The presence of both pro-opiomelanocortin-derived peptides and melanocortin (MC) receptors in nociception-associated areas in the spinal cord suggests that, at the spinal level, the MC system might be involved in nociceptive transmission. In the present study, we demonstrate that a chronic constriction injury (CCI) to the rat sciatic nerve, a lesion that produces neuropathic pain, results in changes in the spinal cord MC system, as shown by an increased binding of (125)I-NDP-MSH to the dorsal horn. Furthermore, we investigated whether intrathecal administration (in the cisterna magna) of selective MC receptor ligands can affect the mechanical and cold allodynia associated with the CCI. Mechanical and cold allodynia were assessed by measuring withdrawal responses of the affected limb to von Frey filaments and withdrawal latencies upon immersion in a 4.5 degrees C water bath, respectively. We show that treatment with the MC receptor antagonist SHU9119 has a profound anti-allodynic effect, suggesting that the endogenous MC system has a tonic effect on nociception. In contrast, administration of the MC4 receptor agonists MTII and d-Tyr-MTII primarily increases the sensitivity to mechanical and cold stimulation. No antinociceptive action was observed after administration of the selective MC3 receptor agonist Nle-gamma-MSH. Together, our data suggest that the spinal cord MC system is involved in neuropathic pain and that the effects of MC receptor ligands on the responses to painful stimuli are exerted through the MC4 receptor. In conclusion, antagonism of the spinal melanocortin system might provide a new approach in the treatment of neuropathic pain.

Hippocampal synaptic plasticity involves competition between Ca2+/calmodulin-dependent protein kinase II and postsynaptic density 95 for binding to the NR2A subunit of the NMDA receptor.

NMDA receptor, Ca(2+)/calmodulin-dependent protein kinase II (alphaCaMKII), and postsynaptic density 95 (PSD-95) are three major components of the PSD fraction. Both alphaCaMKII and PSD-95 have been shown previously to bind NR2 subunits of the NMDA receptor complex. The nature and mechanisms of targeting to the NMDA receptor subunits are, however, not completely understood. Here we report that the C-terminal NR2A(S1389-V1464) sequence was sufficient to guarantee the association of both native and recombinant alphaCaMKII and PSD-95. PSD-95(54-256) was able to compete with the binding of both native and recombinant alphaCaMKII to the NR2A C-tail. Accordingly, alphaCaMKII(1-325) competes with both the native PSD-95 and the native kinase itself for the binding to NR2A. In addition, Ser/Ala1289 and Ser/Asp1289 point mutations on the unique CaMKII phosphosite of NR2A did not significantly influence the binding of native alphaCaMKII and PSD-95 to the NR2A C-tail. Finally, the association-dissociation of alphaCaMKII and PSD-95 to and from the NR2A C-tail was significantly modulated by activation of NMDA receptor achieved by either pharmacological tools or long-term potentiation induction, underlining the importance of dynamic and reciprocal interactions of NMDA receptor, alphaCaMKII, and PSD-95 in hippocampal synaptic plasticity.

The effect of ACTH on rat brain synaptic plasma membrane lipid fluidity.

The effect of ACTH on the lipid fluidity was examined in synaptic plasma membranes from rat forebrain. ACTH1-24 increased the fluidity of the synaptic plasma membranes in a dose-dependent way, the lowest effective dose being 10(-5) M. The shorter N-terminal fragment ACTH1-10 was not effective. The significance of this finding is discussed in relation to the known effects of ACTH on synaptic membrane phosphorylation.

Calcium-dependent turnover of brain polyphosphoinositides in vitro after prelabelling in vivo.

Rat brain phospholipids were labelled in vivo by an intraventricular injection of 32P. The radioactivity was found to accumulate predominantly in limbic structures, particularly hippocampus and diencephalon. A rapid and high specific labelling of the inositol phospholipids and phosphatidic acid was observed. The rate of incorporation into a crude myelin fraction was similar to that into a mitochondrial/synaptosomal fraction although phosphatidyl-myo-inositol 4,5-diphosphate was especially enriched in myelin. Upon incubation in vitro high specific labelling of the inositol phospholipids and phosphatidic acid was observed. The rate of incorporation into a crude myelin fraction was similar to that into a mitochondrial/synaptosomal fraction although phosphatidyl-myo-inositol 4,5-diphosphate was especially enriched in myelin. Upon incubation in vitro high specific labelling of the inositol phospholipids and phosphatidic acid was observed. The rate of incorporation into a crude myelin fraction was similar to that into a mitochondrial/synaptosomal fraction although phosphatidyl-myo-inositol 4,5-diphosphate was especially enriched in myelin. Upon incubation in vitro of the brain fraction after 2 h prelabelling in vivo, both phosphatidyl-myo-inositol 4-phosphate and phosphatidyl-myo-inositol 4,5-diphosphate rapidly lost their radioactivity. Half of the labile fraction of the incorporated 32P was removed within 2 min. None of the other phospholipids changed in the 30 min in vitro incubation period. The metabolism of the polyphosphoinositide proceeded at a lower rate when the temperature was lowered, and was Ca2+-dependent. Further subcellular fractionation revealed that purified synaptosomes and myelin contained highly labelled phosphatidyl-myo-inositol 4-phosphate or phosphatidyl-myo-inositol 4,5-diphosphate. Mitochondria contained highly labelled phosphatidyl-myo-inositol but no phosphatidyl-myo-inositol 4-phosphate or phosphatidyl-myo-inositol 4,5-diphosphate. ACTH1-24 did not inhibit the in vitro dephosphorylation of prelabelled polyphosphoinositide, confirming previous findings that the peptide affects the polyphosphoinositide kinases and not the respective phosphatases.

Adrenocorticotropic hormone (ACTH)-lipid interactions. Implications for involvement of amphipathic helix formation.

ACTH-lipid interactions were investigated by: (1) lipid-monolayer studies using several zwitterionic and anionic phospholipids and gangliosides, (2) permeability experiments by following the swelling rate of liposomes in isotonic glycerol solutions by light scattering, using liposomes of synthetic lipids and liposomes made of lipids extracted from light synaptic plasma membranes, and (3) by steady-state fluorescence anisotropy measurements on liposomes derived from light synaptic plasma membranes employing 1,6-diphenyl-1,3,5-hexatriene as fluorescent probe. (1) The monolayer experiments demonstrated an interaction with gangliosides GT1, GM1, dioleoylphosphatidic acid and phosphatidylserine, but little or no interaction with phosphatidylcholine or sphingomyelin. The interaction with monolayers of GT1 or phosphatidic acid decreased for ACTH1-13-NH2 and ACTH1-10. (2) The liposome experiments showed that 2 X 10(-5) M ACTH1-24 increased the glycerol permeability by 20% and decreased the activation energy only when liposomes derived from light synaptic plasma membranes were used. Treatment of the liposomes with neuraminidase abolished the ACTH-induced permeability increase. (3) Steady-state fluorescence depolarization measurements revealed that ACTH1-24, ACTH1-16-NH2 and ACTH1-10 did not change the fluidity of liposomes derived from light synaptic plasma membranes as sensed by diphenylhexatriene. It is concluded that ACTH1-24 can bind to negatively charged lipids and can form an amphipathic helix aligned parallel to the membrane surface involving the N-terminal residues 1 to 12, possibly to 16. Polysialogangliosides will favorably meet the condition of a high local surface charge density under physiological circumstances. It is suggested that ACTH-ganglioside interactions will participate in ACTH-receptor interactions.

Characterization of phosphatidylinositol-4-phosphate 5-kinase activities from bovine brain membranes.

Phosphatidylinositol-4-phosphate (PtdIns(4)P) kinase activity associated with bovine brain membranes, was released by NaCl treatment and partially purified by chromatography on phosphocellulose, phenylsepharose, Ultrogel AcA44, DEAE-cellulose and ATP-agarose. The final preparation contained a 6333-fold purified protein fraction with a specific activity of 171 nmol.min-1 x mg-1. Under conditions where this PtdIns(4)P kinase activity (PtdIns(4)P kinase activity b) did not bind to DEAE-cellulose, a PtdIns(4)P kinase activity purified earlier (Moritz, A., De Graan, P.N.E., Ekhart, P.F., Gispen, W.H. and Wirtz, K.W.A. (1990) J. Neurochem. 54, 351-354) does bind (PtdIns(4)P kinase activity a). Both enzyme activities specifically used PtdIns(4)P as substrate and phosphorylated the inositol moiety at the 5'-position. PtdIns(4) kinase activity a has an apparent Km of 18 microM for PtdIns(4)P whereas PtdIns(4)P kinase activity b has a Km of 4 microM. All other measured kinetic parameters (i.e., Km for ATP, Mg(2+)-dependence, pH optimum, activation by phosphatidylserine and inhibition by phosphatidylinositol 4,5-bisphosphate) were similar for both enzyme activities.

Beneficial effect of Org 2766 in treatment of peripheral neuropathy in streptozocin-induced diabetic rats.

An injection of streptozocin (STZ) was used to study diabetes-induced peripheral neuropathy in rats. In such rats the values of motor nerve conduction velocity and sensory nerve conduction velocity were decreased compared with the values obtained in nondiabetic controls from 3 wk after STZ injection onward. In recent years it has been extensively documented that peptides related to ACTH and MSH exert a neurotrophic effect on the nervous system that results in enhanced recovery of function after mechanical nerve damage. This article documents the beneficial effect of the peptide Org 2766, an ACTH-(4-9) analogue, in diabetic peripheral neuropathy. Chronic subcutaneous treatment of diabetic rats with Org 2766 results in a significant enhancement of both motor and sensory nerve conduction velocity compared with saline-treated diabetic rats. Histological analysis of cross sections of the sural nerve showed no difference in the total number of nerve fibers in saline- or peptide-treated diabetic rats. In contrast, a difference in fiber size distribution was demonstrated; i.e., the sural nerves of diabetic rats contained fewer thick myelinated fibers. Treatment with Org 2766 resulted in a normal distribution. Apparently, the peptide Org 2766 has a protective action on nerve fibers and nerve function during STZ-induced diabetes.

Place learning and hippocampal synaptic plasticity in streptozotocin-induced diabetic rats.

Moderate impairment of learning and memory has been recognized as a complication of diabetes. The present study examined behavioral and electrophysiological measures of cerebral function in streptozotocin (STZ)-induced diabetic rats. Behavioral testing consisted of a spatial learning task in a water maze. Electrophysiological testing consisted of in vitro assessment of hippocampal long-term potentiation (LTP), an activity-dependent form of synaptic plasticity, which is believed to be related to the cellular mechanisms of learning and memory. Two experiments were performed: the first with severely hyperglycemic rats and the second with moderately hyperglycemic rats. Rats were tested in the water maze 11 weeks after induction of diabetes. Next, LTP was measured in vitro in trained animals. Both spatial learning and LTP expression in the CA1 field of the hippocampus were impaired in severely hyperglycemic rats as compared with nondiabetic controls. In contrast, spatial learning and hippocampal LTP were unaffected in moderately hyperglycemic rats. The association of alterations in hippocampal LTP with specific learning impairments has previously been reported in conditions other than diabetes. Our findings suggest that changes in LTP-like forms of synaptic plasticity in the hippocampus, and possibly in other cerebral structures, are involved in learning deficits in STZ-induced diabetes. The beneficial effect of moderate glycemic control on both place learning and hippocampal LTP supports the significance of the relation between these two parameters and indicates that the development of the observed deficits may be related to the level of glycemic control.

Is the corrected QT interval a reliable indicator of the severity of diabetic autonomic neuropathy?

OBJECTIVE: We investigated whether the corrected QT interval correlated with two other tests for diagnosing autonomic dysfunction in 60 type I diabetic patients with proven peripheral neuropathy. The mean age +/- SD was 48.3 +/- 11.2 yr, the mean duration of diabetes was 24.9 +/- 11.4 yr, and the mean HbA1 was 9.3 +/- 2.4%. RESEARCH DESIGN AND METHODS: All patients underwent three autonomic function tests: 1) the standard five cardiovascular Ewing tests, each scored 0 (normal), 0.5 (borderline), or 1.0 (abnormal). We used the sum of the abnormal findings for the analysis, the cardiovascular autonomic score; 2) measurement of the corrected QT interval taken from a routine electrocardiogram recording; and 3) static and dynamic pupillometry: measurement of dark adapted pupil diameter as percentage of total iris diameter and of pupil constriction latency using an infrared light reflex technique. RESULTS: No significant correlation was found between age, duration of diabetes, or HbA1 and any of the autonomic function tests, except for one between age and cardiovascular autonomic score (r = 0.3202, P = 0.0126). Corrected QT interval did not correlate with cardiovascular autonomic score, pupil diameter, or constriction latency. A significant inverse correlation was found between cardiovascular autonomic score and pupil diameter (r = -0.4861, P < 0.001) and constriction latency (r = 0.3783, P < 0.001). Pupil diameter and constriction latency correlated well (r = -0.4276, P < 0.001). CONCLUSIONS: The corrected QT interval did not correlate with cardiovascular autonomic tests nor pupillometry results. The corrected QT interval therefore should not be used for the diagnosis of the severity of diabetic autonomic neuropathy.

In vivo cardiovascular reactivity and baroreflex activity in diabetic rats.

OBJECTIVES: Abnormalities of the cardiovascular system, e.g. impaired vasoreactivity and changes in baroreflex control of heart rate, are known to occur in experimental diabetes. It is not clear whether these cardiovascular dysfunctions are direct consequences of cardiovascular deficits and/or have autonomic neuropathy as a cause. METHODS: To differentiate between cardiovascular deficits or neuronal impairment as a cause for these cardiovascular dysfunctions, we tested the effects of the ACTH4-9 analogue, Org 2766, a neurotrophic compound without cardiovascular effects, on arterial pressure, heart rate and baroreflex control of heart rate. At 15 weeks, rats were made diabetic by injection of streptozotocin, and from 0-6, 6-12 or 12-18 weeks thereafter 3 groups of rats were treated with Org 2766. These effects were evaluated during phenylephrine-induced increases, and sodium nitroprusside-induced decreases, in blood pressure, in rats that had been diabetic for various periods (2-42 weeks). RESULTS: Throughout, both depressor response and maximal vasodilator activity in response to sodium nitroprusside were significantly (P < 0.05) reduced as compared to those of the non-diabetic controls. The pressor response of the diabetic rats to phenylephrine was only significantly (P < 0.05) reduced at 4, 6 and 12 weeks, and at 18 weeks, the diabetic rats were either hypo- or normoresponsive; Org 2766 did not restore the disturbed pressor response. From weeks 4 to 42 both maximal decrease in heart rate and sensitivity of baroreflex-mediated bradycardia in the diabetic rats were significantly less (P < 0.05) than those in the non-diabetic controls. Org 2766 restored the diminished baroreflex-mediated bradycardia of diabetic rats to non-diabetic control levels at 6 weeks, had an ameliorating effect at 12 weeks and no effect at 18 weeks. CONCLUSIONS: Time-dependent decreases in baroreflex sensitivity in diabetic rats was demonstrated and a much less steep decline of baroreflex sensitivity occurred in non-diabetic control rats. The ACTH4-9 analogue, Org 2766, when given immediately upon the induction of diabetes seem to delay the development of autonomic neuropathy, which suggests that cardiovascular factors appear to be of minor importance.