A Course for Teaching and Learning About the Responsible Conduct of Research.

Over the past 30 years, the scientific community has been increasingly challenged to provide the next generation of researchers with training in responsible conduct of research (RCR). Although RCR courses, workshops, and seminars are now routinely taught internationally, there is little uniformity in goals, content, pedagogy, duration, class size, or methods of assessment. The result is a mixed picture of effectiveness. In this manuscript, we describe goals, rationales, and features for a course tested and revised through well over 100 iterations. Based on our experience and that of others with whom we have shared this model, we propose this course as one that RCR instructors might readily and successfully adopt or adapt.

Effects of hindlimb temperature on sciatic nerve laser Doppler vascular conductance in control and streptozotocin-diabetic rats.

The effects of hindlimb temperature on sciatic nerve and skeletal muscle laser Doppler vascular conductance (LDVC) were assessed in anesthetized control and streptozotocin (STZ)-diabetic rats. With core temperature at 37 degrees C and exposed hindlimb temperature at 32 degrees C, nerve LDVC was significantly lower in rats after 8 weeks of STZ diabetes than in age-matched control rats. Subsequent warming of the exposed hindlimb of control rats from 32 degrees C to 37 degrees C significantly decreased nerve LDVC by 41% and increased muscle LDVC by 48%. Because nerve LDVC was unchanged by hindlimb warming in STZ-diabetic rats, there was no significant difference between control and diabetic nerve LDVC at 37 degrees C. In a second study, after 6 weeks of STZ diabetes, changes from control nerve LDVC were shown to depend on temperature rather than the duration of surgical exposure. These findings emphasize that information about hindlimb temperature is a prerequisite for interpreting the effects of experimental diabetes on hindlimb nerve blood flow.

Histomorphometric analysis of optic nerve changes in experimental glaucoma.

PURPOSE: To assess relative changes in different tissue components of optic nerve and their relationship to nerve fiber loss in the experimental monkey model of glaucoma. METHODS: Chronic intraocular pressure (IOP) elevation was induced by laser trabeculoplasty in the right eye of eight monkeys (Macaca fascicularis). Both experimental right optic nerves and control left optic nerves were studied. Histomorphometric analysis was performed on optic nerve cross-sections using bright field microscopy with camera lucida. Cross-sectional areas of optic nerve tissue components were estimated by point counting. Nerve fiber density was estimated by unbiased random sampling. Nerve fiber number was calculated by multiplying nerve fiber density with neuroglial area. RESULTS: Varying degrees of nerve fiber loss were seen in eight optic nerves with chronic IOP elevation. More than 50% nerve fiber loss was noted in four of eight experimental optic nerves. In these severely affected optic nerves, total optic nerve area was significantly decreased compared with control optic nerves. Among the optic nerve tissue components, only the ratio of myelinated fiber area to total optic nerve area was significantly decreased. The ratio of extraaxonal area to total optic nerve area was significantly increased, whereas the ratio of interfascicular septal area to total optic nerve area did not change significantly. For all optic nerves, differences in nerve fiber count between control and experimental optic nerves showed the strongest correlation with differences in myelinated fiber area, followed by differences in extraaxonal area and total optic nerve area. CONCLUSION: This histomorphometric study suggests the validity of the experimental monkey model of glaucoma in studying changes occurring in the nonaxonal optic nerve tissue components in human glaucomatous optic neuropathy. Glial scar tissue area was significantly increased in optic nerves with severe glaucomatous damage. Although a decrease in total optic nerve area was observed, among the optic nerve tissue components only myelinated nerve fiber area decreased significantly. Myelinated nerve fiber area also showed the strongest association with nerve fiber loss in experimental glaucoma.

Classification of teased nerve fibers for multicenter clinical trials.

Teased nerve fibers are used widely in both clinical and experimental neuropathology, but anecdotal evidence indicates that even experienced readers find little agreement on categories for teased fiber classification. To develop a classification scheme that could be used and understood by both experienced and naive readers, specific criteria were developed for normal fibers and those exhibiting Wallerian degeneration, demyelination, hypomyelination, remyelination, and abnormal paranodal myelination. Twenty fibers teased from human sural nerve biopsies were selected as examples of one or more of these categories. Ten readers, including seven having no previous experience with teased fibers, were given a set of instructions and asked to score each fiber for all matching categories. These readers averaged high rates of true positive (56-85%) classifications, while average false positive (3-18%) rates were much lower. Among the three experienced readers, true positive agreements averaged between 75 and 100% across the fiber classifications. False positives were correspondingly low, ranging between 0 and 8%. These results suggest that it is possible to design an easily learned, meaningful scheme for classifying teased nerve fibers.

Nerve conduction velocity, laser Doppler flow, and axonal caliber in galactose and streptozotocin diabetes.

In an initial study, the effects of galactose intoxication on nerve laser Doppler blood flow (NLDF) and nerve conduction velocity (NCV) were assessed after 1-16 weeks of galactose feeding in pentobarbital-anesthetized rats. NLDF was not significantly changed at any time point. NCV was significantly reduced after 16, but not 1 or 4, weeks of galactose feeding. In a second study, NLDF was not significantly changed by 4 weeks of galactose intoxication, but streptozotocin-diabetic NLDF was significantly reduced compared to both control (P<0.001) and galactose-intoxicated rats (P<0.05). Compared to control animals, sciatic motor NCV was significantly (P<0.001) reduced in the galactose group, while sciatic and saphenous sensory NCVs were not significantly changed. In the streptozotocin-diabetic rats, motor and sensory NCVs were all significantly reduced (P<0.001). In contrast to the NCV findings, mean caliber of myelinated axons in both the saphenous and sciatic nerves was reduced in galactose-intoxicated, but not streptozotocin-diabetic rats. The observed sequence of changes associated with these two models of diabetic neuropathy is not consistent with the proposed roles of ischemia and axonal dwindling in the reported nerve conduction deficits.

NT-3 attenuates functional and structural disorders in sensory nerves of galactose-fed rats.

The present study investigated the effect of NT-3, a neurotrophin expressed in nerve and skeletal muscle, on myelinated fiber disorders of galactose-fed rats. Adult, female Sprague-Dawley rats were fed diets containing complete micronutrient supplements and either 0% D-galactose (control) or 40% D-galactose. Treated controls received 20 mg/kg NT-3 and treated galactose-fed rats received 1, 5, or 20 mg/kg NT-3 three times per week by subcutaneous injections. After 2 months, sciatic and saphenous sensory nerve conduction velocity (SNCV) and sciatic motor nerve conduction velocity (MNCV) were measured and the sciatic, sural, peroneal and saphenous nerves and dorsal and ventral roots processed for light microscopy. Treatment of control animals with NT-3 had no effect on any functional or structural parameter. Compared to control values, galactose feeding induced a sensory and motor nerve conduction deficit and a reduction in axonal caliber. Treatment with 5 and 20 mg/kg NT-3 ameliorated deficits in sciatic and saphenous SNCV in galactose-fed rats but had no effect on the MNCV deficit. NT-3 treatment also attenuated the decrease in mean axonal caliber in the dorsal root and sural nerve but not in the saphenous nerve, ventral root and peroneal nerve. These observations show that NT-3 can selectively attenuate the sensory conduction deficit of galactose neuropathy in a dose-dependent manner that depends only in part on restoration of axonal caliber of large-fiber sensory neurons.

Tactile hyperesthesia, altered epidermal innervation and plantar nerve injury in the hindfeet of rats housed on wire grates.

The effects of wire grates on nerve injury and recovery were examined in rats housed in cages with sawdust-covered solid flooring. For the first 3 weeks of the study, 20 rats were housed on sawdust alone and 20 rats were housed in cages with wire grates placed over the sawdust. For the remaining 9 weeks, 10 animals housed on sawdust had wire grates added to their cages, while grates were removed from the cages of 10 animals. The effects of tactile stimulation on hindpaw plantar skin was measured weekly using the Von Frey filament test. Intraepidermal innervation using PGP 9.5 immunostaining and plantar nerve histology were assessed at the end of the 12-week study. After just 1 week on grates, hindpaw withdrawal thresholds were already markedly decreased and remained low until the grates were removed at 3 weeks. Thresholds returned to normal by 4 weeks after removal of the grates. Wire grates also induced increases in PGP 9.5 immunoreactive intraepidermal fine nerve endings that were normalized after grate removal. Demyelination, Wallerian degeneration and Renaut bodies were induced in the medial plantar nerve in rats housed in cages with wire-grate flooring. Nerve injury was largely resolved after 9 weeks on sawdust flooring. These data demonstrate that wire grates rapidly induce hindpaw tactile hyperesthesia and plantar neuropathy in rats and emphasize a risk of using wire-grate cage flooring in studies assessing hindlimb function and structure.

Relationship of optic disc topography to optic nerve fiber number in glaucoma.

OBJECTIVE: To assess the relationship between in vivo measurements of optic disc topography and histomorphometric measurements of optic nerve fiber number in glaucoma. METHODS: Both eyes of 10 monkeys (Macaca fascicularis) with laser-induced glaucoma in the right eye were studied. Optic disc topography was measured in vivo with a confocal scanning laser ophthalmoscope. Histomorphometry was performed on optic nerve cross sections using bright-field microscopy with camera lucida. Nerve fiber density was estimated by unbiased random sampling. Nerve fiber number was estimated for each sector by multiplying nerve fiber density with neuroglial area. Nerve fiber count was compared with each of 13 global optic disc topographic parameters. RESULTS: For neuroretinal measurements in the glaucomatous eyes, rim area, retinal nerve fiber layer (RNFL) cross-sectional area, rim volume, and RNFL thickness correlated significantly with optic nerve fiber number. Differences in nerve fiber count between control and glaucomatous optic nerves showed the strongest correlation with differences in mean height contour; this was followed by RNFL cross-sectional area, RNFL thickness, rim volume, and differences in rim area. For cup measurements in the glaucomatous eyes, cup volume below reference, cup area, mean cup depth, the ratio of cup area to disc area, and cup shape correlated significantly with nerve fiber number. Differences in nerve fiber number between control and glaucomatous optic nerves showed the strongest correlation with differences in cup shape; this was followed by mean cup depth, cup volume below reference, the ratio of cup area to disc area, cup area, and differences in cup volume below surface. No association was found between optic nerve fiber number and optic disc area in glaucomatous eyes. CONCLUSIONS: In experimental glaucoma, most optic disc topography measures correlated significantly with optic nerve fiber number. The results of this histomorphometric study support the use of confocal scanning laser ophthalmoscopy to evaluate optic nerve damage in glaucoma.

Reactive, degenerative, and proliferative Schwann cell responses in experimental galactose and human diabetic neuropathy.

Despite early descriptions of hypertrophic Schwann cells and onion-bulb formation in patients with diabetic neuropathy, clinical and experimental studies have emphasized axonal pathology. In recent years, the Schwann cell has been further implicated in diabetic neuropathy because it is the primary intrafascicular location for the first enzyme of the polyol pathway, aldose reductase, which appears to have a role in modulating a variety of complications of diabetes, including diabetic neuropathy. To further explore the role of polyol pathway flux in the pathogenesis of Schwann cell injury, ultrastructural abnormalities of Schwann cells in human diabetic neuropathy (HDN) were compared with those in experimental galactose neuropathy (EGN), a well-characterized model of hyperglycemia without hypoinsulinemia. Similar to previous studies of EGN, reactive, degenerative and proliferative changes of Schwann cells were observed after 2, 4 and 24 months of galactose intoxication. Reactive changes included accumulation of lipid droplets, pi granules of Reich and glycogen granules, increased numbers of subplasmalemmal vesicles, cytoplasmic expansion, and capping. Degenerative changes included enlargement of mitochondria and effacement of cristae, and disintegration of both abaxonal and adaxonal cytosol and organelles. Both demyelination and onion-bulb formation were seen at all time points, although supernumerary Schwann cells and axonal degeneration were most numerous after 24 months of galactose feeding. In sural nerve biopsy samples from patients with diabetes and progressive worsening of neuropathy, ultrastructural abnormalities in Schwann cells encompassed the full range of reactive, degenerative and proliferative changes described in galactose-fed rats. The concordance of fine-structural observations in nerves from galactose-fed rats and these adult-onset diabetic patients emphasizes the role of flux through aldose reductase in the complex pathology of diabetic neuropathy and points to the utility of galactose intoxication in helping to understand this metabolic disorder.

Effects of body and hindlimb temperature on laser Doppler blood flow and vascular conductance in rat sciatic nerve and skeletal muscle.

The role of body and hindlimb temperature in the control of blood flow in nerve and muscle was assessed by laser Doppler flowmetry. Following surgical exposure of nerve, initial measurements were taken for 5 min at hindlimb temperatures of 30-31 degrees C. A second set of identical measurements was then made either with or without warming to 37 degrees C. Without warming, nerve laser Doppler flow (NLDF) increased by 14.5+/-3.2% (mean+/-SEM) but, with warming, decreased by 40.9+/-8.2%. In contrast, adduccamerontor magnus muscle laser Doppler flow (MLDF) decreased without warming (14.7+/-1.0%) and increased with warming (20.4+/-12.8%). Because blood pressure was not significantly altered by changes in hindlimb temperature, the patterns of changes in vascular conductance (laser Doppler flow/blood pressure) were not significantly different from that seen with NLDF and MLDF. Thus, warming to physiological temperatures was associated with decreased NLDF and vascular conductance and increased MLDF and vascular conductance. These data may have implications for the interpretation of nerve blood flow data obtained at different hindlimb temperatures.

Effects of aging on nerve conduction block induced by bupivacaine and procaine in rats.

To test the hypothesis that the dose requirement for local anesthetics is changed in aged animals, the effects of two different local anesthetics on nerve conduction block were tested in young and old rats. Young (6 months) and old (27 months) male Fisher-344 rats were anesthetized with intraperitoneal pentobarbital and diazepam. Stimulating electrodes were placed in the sciatic notch and in the ankle and recording electrodes were placed distally in the ipsilateral foot to record evoked electrical activity of the interosseous muscles. Motor nerve conduction velocity was significantly less in old (48.8 +/- 3.9, mean +/- SD m.sec-1) than in young rats (56.4 +/- 10.3 m.sec-1) (P < 0.05). To test the effects of aging on conduction block, equipotent doses of bupivacaine (0.2%), an amide-linked local anesthetic, or procaine (0.6%), an ester-linked local anesthetic, were injected next to exposed sciatic nerves and evoked electrical activity was monitored following repeated stimulation at the sciatic notch. At 10 minutes after injection, bupivacaine produced significantly greater nerve block in old rats (100 +/- 0.0%) than young rats (29.8 +/- 41.6%) (P < 0.01); the difference for procaine (old 67.5 +/- 40.4% vs. young 30.4 +/- 35.3%) was not statistically significant. The lower dose requirement for bupivacaine, and the apparent differences compared to procaine, may have implications for the use of local anesthetics in an aging patient population.

Effects of seven days of galactose feeding and aldose reductase inhibition on mast cells and vessel morphometry in rat sciatic nerve.

The association between mast cells and vessel morphometry in sciatic nerve was examined after seven days in animals fed a diet of 40% D-galactose and compared to control rats and to galactose-fed animals treated with the aldose reductase inhibitor, Tolrestat. Electron microscopy revealed an increase in the total number of mast cells and the number of degranulated mast cells in galactose-fed animals (7.8 +/- 2.9; 2.6 +/- 2.9; mean +/- SD) compared to controls (4.6 +/- 2.1; degranulated mast cells were not seen in any control nerves) and Tolrestat-treated, galactose-fed animals (4.4 +/- 2.5; 0.1 +/- 0.4). Although no significant differences were noted in the numbers of vessels between the three groups, an index of vasoconstriction was significantly increased in the galactose-fed animals (0.115 +/- 0.048; mean +/- SD) compared to controls (0.068 +/- 0.011) and Tolrestat-treated, galactose-fed animals (0.075 +/- 0.20). These data suggest that mast cell degranulation is associated with the vascular constriction induced by seven days of galactose intoxication and that both may be prevented by inhibiting aldose reductase.

Polyol pathway and osmoregulation in JS1 Schwann cells grown in hyperglycemic and hyperosmotic conditions.

To test the osmoregulatory rules of Schwann cell aldose reductase (AR) and myo-inositol, JS1 Schwann cells were grown under control and hyperosmotic conditions with and without excess glucose or galactose. JS1 cells cultured in control conditions possessed AR protein and activity that were not altered by the inclusion of 25 mM glucose or galactose. Following culture with 100 mM NaCl, there was a decline in cell number accompanied by an increase in AR activity, both of which were attenuated by the addition of 25 mM glucose or galactose. Sorbitol was not detected in JS1 Schwann cells following culture in control, glucose-supplemented, or hyperosmotic medium, and dulcitol accumulated only following culture with galactose. However, both polyols were dramatically increased in JS1 cells cultured in hyperosmotic medium supplemented with 25 mM glucose or galactose. In contrast, myo-inositol levels were elevated only during hyperosmotic exposure but decreased when glucose or galactose was also present. These data are consistent with the use of polyol formation by JS1 Schwann cells as a means of responding to osmotic stress.

Mast cell degranulation and blood-nerve barrier permeability in rat sciatic nerve after 7 days of hyperglycemia.

The association between hyperglycemia and altered blood-nerve barrier permeability was examined after 7 days of experimental diabetes. In nerves of rats fed a diet of 40% galactose, permeability to [14C]mannitol [13.43 +/- 2.47 x 10(-5) (SD) ml.s-1.g dry wt-1] and water content [3.43 +/- 0.24 (SD) mg/mg dry wt] were significantly increased compared with control (9.24 +/- 2.09 x 10(-5) ml.s-1.g dry wt-1 and 2.15 +/- 0.28 mg/mg dry wt) and streptozotocin-diabetic animals (8.43 +/- 2.94 x 10(-5) ml.s.-1.g dry wt-1 and 2.35 +/- 0.56 mg/mg dry wt). Electron microscopy revealed significant increases in the number of degranulating perivascular mast cells and in an index of vasoconstriction in galactose-treated rats (3.8 +/- 1.6 and 0.160 +/- 0.062, respectively) compared with control (0.5 +/- 0.8 and 0.072 +/- 0.017, respectively) and diabetic animals (1.4 +/- 1.7 and 0.083 +/- 0.033, respectively). The data are consistent with a role for mast cells in permeability changes occurring after only 7 days of galactose intoxication.

Effects of cocaine on blood flow and prostaglandin metabolites in rat sciatic nerve.

To better understand the mechanisms of local anesthetic-reduced nerve blood flow and nerve blood flow regulation, the effects of cocaine on blood flow and vasoactive prostaglandins were tested in the sciatic nerve of anesthetized rats. After 30 min, nerve blood flow was significantly reduced from baseline by perineural injection of 160 mM cocaine [-29.4 +/- 4.0 (SD) laser-Doppler flow units (P < 0.001)] but not saline (1.6 +/- 11.3). These same nerves were removed and assayed for the stable metabolites 6-ketoprostaglandin F1 alpha and thromboxane B2 of the vasoactive eicosanoids prostacyclin and thromboxane A2, respectively. Both metabolites were reduced, but the ratio of thromboxane B2 to 6-ketoprostaglandin F1 alpha was greater (P < 0.05) in nerves pretreated with cocaine (6.1 +/- 3.2 vs. 2.4 +/- 1.1) and was inversely correlated (P < 0.01) with nerve blood flow. In a separate experiment, perineural injection of the prostaglandin synthesis inhibitor indomethacin (0.5 mg in 0.5 ml of either saline or 50% ethanol) reduced nerve blood flow as well (P < 0.05). These results are consistent with the proposal that cocaine inhibits nerve blood flow by effects on nerve prostaglandin metabolites.

Aldose reductase inhibition, Doppler flux and conduction in diabetic rat nerve.

Two chemically distinct aldose reductase inhibitors, ponalrestat and tolrestat, were tested against laser Doppler blood flow and conduction deficits in the sciatic nerve of diabetic rats. The effects of two months of streptozotocin-induced diabetes and aldose reductase inhibition on body weight, plasma glucose, and nerve sugars and polyols were comparable to those reported previously. Nerve blood flow, reflected by laser Doppler flow measurements, and motor nerve conduction velocity were both significantly less in diabetic than in control animals. Both of these reductions were prevented by ponalrestat, but not tolrestat. Thus, either deficits in laser Doppler blood flow and conduction are not aldose reductase inhibitor-dependent or tolrestat has some other property which offsets the beneficial effects of aldose reductase inhibition. In either case, these data are consistent with the hypothesis that reduced nerve blood flow contributes to conduction deficits in diabetes.

Physiologic mechanisms by which local anesthetics may cause injury to nerve and spinal cord.

OBJECTIVE: Review evidence for possible physiologic mechanisms by which local anesthetics cause nerve injury was reviewed. METHODS: Published clinical case reports, and both in vivo and in vitro experimental studies in animals, were reviewed. Reports with direct bearing on possible mechanisms of local anesthetic neurotoxicity were included. RESULTS: There is basic and clinical evidence that local anesthetics can cause nerve injury. Nerve injury may result from direct toxicity to the axon or Schwann cell, or may be secondary to disruption of the nerve microenvironment. Although each of these possibilities is supported by published experiments, injury to the axon is the most likely explanation for persistent neurologic deficits, such as those believed to result from clinical use of local anesthetics. Whether the injury is direct or indirect, and the precise mechanism by which it occurs, has received only limited study. CONCLUSIONS: In rare instances, the clinical use of local anesthetics is associated with neurologic morbidity. The physiologic mechanisms of toxicity for which there is the best evidence are inhibition of fast axonal transport, disruption of the axonal cytoskeleton, axonal degeneration, and ischemic nerve injury. The effects of local anesthetics on nerve blood flow may be related to inhibition of endothelium-dependent vasodilation or interruption of the synthesis of vasodilating prostaglandins. Both the prevention and the treatment of nerve injury caused by local anesthetics requires information that is not yet available about the physiologic and molecular mechanisms for direct neural toxicity of local anesthetics.

Permeability and surface area of the blood-nerve barrier in galactose intoxication.

The blood-nerve movement of a small molecular weight non-electrolyte was studied in control and galactose-fed rats by measuring the permeability-surface area (PSA) product of the blood-nerve interface to [14C]mannitol in sciatic nerve using an in vivo injection method. PSA products were measured after 9 to 11 months of feeding control rats a diet containing 0% galactose and galactose-intoxicated rats a diet containing 40% galactose. Nerves of the galactose-fed group were hydrated as reflected by a significant increase in nerve water content and wet weight to dry weight ratio (both P < 0.05). Compared to controls, PSA products were increased by 51% (P < 0.01) in galactose-fed animals when referenced to nerve dry weight (13.59 +/- 2.90 x 10(-5) ml/s/g dry wt. versus 8.99 +/- 1.59 x 10(-5) ml/s/g dry wt.; mean +/- S.D.; galactose vs. control, respectively) or by 30% (P < 0.001) when referenced to nerve length (2.43 +/- 0.43 x 10(-5) ml/s/mm vs. 1.87 +/- 0.48 x 10(-5) ml/s/mm) but not when referenced to nerve wet weight. It is suggested that in galactose intoxication, where endoneurial volume changes reflect increases in nerve water content, PSA products are best normalized to dry weight or length, which are not affected by volume changes. Normalized to dry weight, the blood-nerve barrier surface area (i.e. vessels and perineurium) was determined by morphometric methods to be increased by 34% in the galactose-intoxicated group.(ABSTRACT TRUNCATED AT 250 WORDS)

Relative neural toxicity of local anesthetics.

In rat sciatic nerve, relative neural toxicity and relative motor nerve conduction blockade were assessed for two amide-linked local anesthetics (etidocaine and lidocaine) and two ester-linked local anesthetics (chloroprocaine and procaine). As measures of neural toxicity, nerve fiber injury and edema were assayed by light microscopic examination of nerve tissue sampled 2 days after perineural (next to the sciatic nerve) injection of various concentrations of the local anesthetics. Both nerve injury and edema increased with concentration of local anesthetics, but injury was frequently present in nerve fascicles with little or no edema. In parallel studies, the amplitude of the electrical activity elicited from the interosseous muscles of the foot following ipsilateral electrical stimulation at the sciatic notch was monitored for up to 15 minutes to assess the extent of motor nerve blockade. The resulting log concentration-response curves were analyzed for differences in potency. Both for injury and for conduction block, the order of decreasing potency was: etidocaine, lidocaine, chloroprocaine, procaine. These results are not consistent with the proposal that ester-linked agents are more likely than other local anesthetic agents to cause nerve injury.