James M. Kerns

Effects of pudendal nerve injury in the female rat

To test a neurogenic hypothesis for external urethral sphincter (EUS) dysfunction associated with urinary incontinence, the proximal pudendal nerve was crushed in anesthetized retired breeder female rats (n = 5) and compared with a sham lesion group (n = 4). Outcome measures included concentric needle electromyograms (EMGs) from the target EUS, voiding patterns during a 2‐hour dark period, and micturition data over a 24‐hour period. Fast Blue (FB) was introduced to the crush site at the time of injury and Diamidino Yellow (DY) to the EUS at the time the rats were killed (3 months post‐operative), when histological analysis of the nerve and urethra was also performed. EMG records indicated the EUS motor units undergo typical denervation changes followed by regeneration and recovery. Voiding patterns from the crush group show a significant increase of small urine marks in the front third of the cage. At 1–2 weeks post‐op, the frequency of voids was significantly increased in the crush group compared to pre‐op and late post‐op time periods. The mean volume voided in the light phase at the early post‐op time was significantly increased in the sham group. Light and electron microscopic patterns seen in nerve and muscle suggest the regenerating motor units maintain a structural integrity. Motoneurons in the lower lumbar cord were labeled with either DY (14.5 ± 6.8), FB (31.7 ± 23.7), or both (35.0 ± 17.5) tracers, indicating ∼54% of the crushed pudendal neurons regenerated to the EUS. In conclusion, several measures suggest this reversible crush lesion induces mild urinary incontinence. This animal model is promising for further development of hypotheses regarding neural injury, the pathogenesis of incontinence, and strategies aimed at prevention and treatment. Neurourol. Urodynam. 19:53–69, 2000.

Characterization of a new animal model for evaluation of persistent postthoracotomy pain

Chronic pain after thoracotomy is common, although its basis and therapy have not been well characterized. In this study we characterize the allodynic responses (mechanical and cold) as well as the histopathologic changes after thoracotomy and rib retraction in rats. The antinociceptive effect of systemic and intrathecal analgesics was also evaluated. Male Sprague-Dawley rats were anesthetized and the right 4th and 5th ribs surgically exposed. The pleura was opened between the ribs and a retractor placed under both ribs and opened 8 mm. Retraction was maintained for 5, 30, or 60 min. Control animals had pleural incision only. Beginning Day 2 postsurgery, animals were tested for mechanical allodynia using calibrated von Frey filaments and cold allodynia using acetone applied to the incision site. Two weeks after surgery, animals were tested for reduction of allodynia with intraperitoneal and intrathecal injections of analgesics. Intercostal nerve histology was examined at 14 days postsurgery. Allodynia developed in 50% of the animals with 60 min retraction but in only 11% and 10% of animals when the retraction time was 5 and 30 min, respectively, and in none of the control animals. Allodynic animals showed extensive axon loss in the intercostal nerves of the retracted ribs. Allodynia appeared by Day 10 in the rib-retraction model and lasted at least 40 days. Systemic morphine sulfate (50% effective dose [ED50], 1.06 mg/kg) and gabapentin (ED50, 24.2 mg/kg), as well as intrathecal morphine (ED50, 1.19 nmol), gabapentin (ED50, 13.8 nmol), clonidine (ED50, 72.7 nmol), and neostigmine (ED50, 0.54 nmol) reduced allodynia. Rib-retraction in rats for 60 min produces allodynia that lasts more than 1 mo, and this allodynia is reduced by morphine, gabapentin, clonidine, and neostigmine. This new model may be useful for quantifying the efficacy of techniques to reduce the frequency and severity of long-term postthoracotomy pain.

The influence of predegeneration on regeneration through peripheral nerve grafts in the rat

Nerve regeneration through predegenerated (PNG) or fresh (FNG) autografts in either fresh or delayed recipient nerve beds were studied in the rat sciatic nerve. Grafts 10 mm in length were excised either immediately or following a 7-day period of predegeneration. They were sutured into gaps on the contralateral side which were either freshly made or had been made 7 days previously. The early recovery (10 days at 2-day intervals) was evaluated by the sensory pinch test to measure the rate of regeneration and the delay period. The PNG group had an improvement in axonal regeneration as evidenced by a reduced delay period, a reduced number of regeneration failures, and less variability in regeneration distances compared to the FNG group. Conditioning the host site blocked some of the delay reduction in the PNG group, but had no effect on regeneration rate or delay in the FNG group. The presence of axons in the graft was confirmed by immunocytochemistry for neurofilament protein and by electron microscopy. The results suggest that proliferated cells, primarily Schwann cells, promote regeneration through the suture line and graft into the distal segment.

Pre-degenerated nerve grafts enhance regeneration by shortening the initial delay period.

In the present study we tested how nerve grafts with different pre-degeneration periods (1-28 days) influenced the early regenerative response in the rat sciatic nerve. The sciatic nerve on the right side was crushed and after 1-28 days of pre-degeneration, a 10 mm segment was used as an autologous nerve graft and transposed to a freshly made 10 mm long nerve defect on the left side. The regeneration distance was measured by the sensory pinch test 2-10 days after nerve repair. A newly developed mathematical model was used to calculate regeneration rates and initial delay periods from the measured regeneration distances. Pre-degenerated nerve grafts improved nerve regeneration by decreasing the initial delay period as compared to fresh nerve grafts without affecting the regeneration rate. Only one day of pre-degeneration was sufficient to reduce the initial delay period from 3.6 days to 1.7 days. The maximal effect on the initial delay period was achieved after 3 days of pre-degeneration. The initial delay period at later pre-degeneration intervals (7-14 days) was about 1 day. The effect persisted for at least 28 days of pre-degeneration. The regeneration rate was 1.5 mm/day for fresh nerve grafts and between 1.8-2.1 mm/day for pre-degenerated grafts. The results suggest that the effects of pre-degeneration are not only due to the increased cell proliferation in the graft, but that also trophic and/or inflammatory mechanisms may be of importance. Grafts pre-degenerated by crush may have clinical implications since they are easy to perform if an elective nerve grafting procedure is planned.

Predegeneration enhances regeneration into acellular nerve grafts

In the present study, we determined the regeneration rate and the initial delay in rat sciatic nerve grafts first made hypercellular by predegeneration then acellular by freeze-thawing. 7-day predegenerated nerve pieces from the distal nerve stump on the right side were made acellular by repeated freeze-thawing and inserted as grafts into a 10-mm long freshly created defect on the left contralateral side. Freshly made (no predegeneration period) acellular nerve grafts were used as controls. Both types of grafts supported outgrowth of regenerating axons as demonstrated by the sensory pinch test. However, the predegenerated acellular nerve grafts had a significantly shorter initial delay period (2.7 days) as compared with freshly made acellular nerve grafts (9.5 days). The initial delay period for predegenerated acellular nerve grafts was similar to that for fresh cellular nerve grafts but significantly longer than that for predegenerated cellular nerve grafts [24]. The rate of regeneration appeared independent of the type of grafts used. We suggest that modifications of the basal lamina and/or factors produced during the predegeneration period by non-neuronal cells survive the freeze-thawing cycle and account for the decrease in the initial delay period.

Local anesthetic sciatic nerve block and nerve fiber damage in diabetic rats

Background and Objectives: A concern for anesthesiologists is whether local anesthetics are more toxic to peripheral nerves in diabetic patients. A previous study in streptozotocin-induced diabetic rats showed that larger doses of lidocaine produce moderate nerve injury after nerve block in normal rats and worse injury in diabetic rats. However, it is not clear whether a smaller local anesthetic dose that produces negligible nerve fiber damage in normal rats will produce significant nerve damage in diabetic rats and if adding adjuvant drugs modulates this effect. Methods: Rats were intravenously injected with 50 mg/kg streptozotocin to induce diabetes (blood glucose levels >250 mg/dL) and diabetic neuropathy. After waiting 35 days, an injection (0.1 mL) of 1% lidocaine alone, or with 5 &mgr;g/mL epinephrine or 7.5 &mgr;g/mL clonidine added, or 0.5% ropivacaine alone was performed at the left sciatic notch in both diabetic and nondiabetic rats. The duration of sensory (pin prick) and motor (toe spreading reflex) nerve block in the hind paws was determined. For histologic controls, all rats also received saline vehicle injection into the right sciatic notch. Another group of uninjected rats was used as naive controls. Left and right nerves were removed 2 days after injection and fixed in situ with a 4% glutaraldehyde solution. Myelinated axon profiles suggestive of neuropathy (myelin figures, pale and swollen, or dark-staining axoplasm) were counted and expressed as a percentage of the total number of fibers in each rat sciatic nerve. Results: All streptozotocin-injected rats became diabetic and had pronounced tactile allodynia. All rats had sensory and motor nerve blocks lasting for at least 50 mins after injection of local anesthetic. The duration of sensory and motor nerve block was longer in diabetic rats than in nondiabetic rats for all drug groups tested. None of the sciatic nerves examined showed greater than 3% nerve fiber degeneration. Although lidocaine in diabetic rats did not produce nerve fiber damage, diabetic rats receiving lidocaine/clonidine or ropivacaine had more abnormal myelinated axon profiles than did nondiabetic rats receiving the same drug. Conclusions: The duration of sciatic nerve block with local anesthetics is longer in diabetic compared with nondiabetic rats. A small, but statistically significant, increase in nerve damage occurred in diabetic rats after nerve block with ropivacaine alone or when duration of lidocaine block was extended with clonidine. These findings may have implications for dosing of local anesthetics in diabetic patients undergoing regional analgesia with nerve blocks.

A comparison of cryoprobe and crush lesions in the rat sciatic nerve.

&NA; This study examines the behavioral, sensory, motor and structural recovery during the first 2 months following a freeze (cryoprobe) lesion compared to a nerve crush (forceps). There is a complete loss of sensory and motor function following either type of lesion during the first 2 weeks of recovery. The toe spreading reflex and the sciatic functional index of locomotion behavior returned to normal without significant group differences. Latency times for the pain withdrawal reflex were slightly shorter in the cryoprobe group, but both groups returned to baseline during the second month. An improved regenerative pattern was suggested for the motor recovery in the cryoprobe group as expressed by the amplitude of the digital twitch tension curves. However, the respective curve areas were not different. Morphometric analysis indicated a significant reduction in the distal nerve cross‐sectional area, an increase in the mean myelinated fiber density and an increase in the estimated total number of myelinated nerve fibers in both experimental groups. Mean fiber diameter and myelin sheath thickness had not fully returned to normal in either experimental group. Both the fiber size and myelin sheath thickness were significantly reduced in the cryoprobe group. In conclusion, the two lesion types have remarkably similar patterns of recovery. Functional data suggest that motor recovery precedes sensory recovery following a cryoprobe lesion.

The effect of chronic subarachnoid bupivacaine infusion in dogs

A functional and histologic examination of the effect of the continuous intrathecal administration of bupivacaine was made in five dogs. After a partial laminectomy at L5, a silicone rubber catheter was inserted into the subarachnoid space and advanced 10 cm cranially. A model 400 Infusaid pump, used for drug delivery, was placed in a subcutaneous pocket between the 13th rib and iliac crest. Drug infusions were individually adjusted and maintained at a concentration such that the animal just exhibited slight gait impairment. Daily bupivacaine doses ranged from 5.7–11.1 mg. Infusions were maintained for a period of 3–16 weeks. Light microscopic examination of spinal cord and roots revealed no abnormalities. A focal mononuclear cell infiltration of the leptomeninges was seen in two drug animals, as well as one catheter control animal. One of the 16-week animals had a residual limp upon drug removal. While we were not able to quantitatively assess the degree of sensory motor dissociation, the results of this study suggest that chronic intrathecal bupivacaine infusion through an implantable pump system may be a short-term alternative to intrathecal morphine in the control of cancer pain.

Effect of repetitive lidocaine infusion on peripheral nerve

Neurotoxicity in rat tibial nerve was assessed following injections of lidocaine through implanted catheters. Doses of 1 to 4% lidocaine in volumes sufficient to inhibit motor nerve function, were infused three times a day for 3 days. When 4% lidocaine was infused into a Silastic cuff surrounding the sciatic nerve, all function was lost and there was considerable nerve fiber degeneration. When 4% lidocaine was infused within the muscle pocket surrounding the nerve, but at some distance from the nerve, there was no measurable permanent loss of motor function, but occasional nerve fiber degeneration. In contrast, 1% lidocaine appeared to cause no neurotoxicity, even when infused within a surrounding cuff. In addition, 1% lidocaine injection did not cause damage to the underlying muscle. Therefore, intermittent nerve block with low concentrations of lidocaine appears to be nontoxic to nerve and muscle when administered during 3 days. However, long-term studies are needed before clinical usage can be considered, because there is the potential for nerve and muscle damage.

Striated Muscle and Nerve Fascicle Distribution in the Female Rat Urethral Sphincter

The anatomical basis for urinary continence depends on a thorough understanding of the tissues in the urethra. The objective of this study was to evaluate the morphology and neuroanatomy of urethral striated muscle, called the rhabdosphincter or external urethral sphincter, in normal female rats. Urethras from 12 female rats were dissected from the bladder, fixed, embedded in paraffin or epon, and sectioned every 1 mm. Striated muscle content was taken as the ratio of the striated muscle area to net urethral area. Nerve fascicles containing myelinated axons near the rhabdosphincter were counted and mapped. Both striated muscle content and number of nerve fascicles peak in the proximal third of the urethra, with a secondary peak at the distal end of the urethra. This secondary peak may correspond to an analog of the combined compressor urethrae/urethrovaginal sphincter located in the distal urethra in human. The rhabdosphincter has a variable distribution along the length of the urethra. In the middle and distal thirds of the urethra, the dorsal striated muscle fibers between the urethra and vagina become more sparse. The majority of nerve fascicles are contained in the lateral quadrants of the urethra, similar to the lateral distribution of somatic nerves in humans. In conclusion, this study demonstrates the normal distribution of the striated musculature and neuroanatomy in the urethra, with similarities to the human. It thus supports and extends the usefulness of the rat as an experimental model for studying urinary incontinence. Anat Rec 290:145–154, 2007.