Characterization of nerve growth factor-induced mechanical and thermal hypersensitivity in rats.

BACKGROUND: Injection of nerve growth factor (NGF) produces mechanical and thermal hypersensitivity in rodents and humans. Treatment with sequestering antibodies demonstrates the importance of NGF in various pain states, with efficacy seen in a number of animal pain models and in painful human conditions. However, these phenomena have not been evaluated in the context of using NGF-induced hypersensitivities as a model of pain. METHODS: NGF-induced behaviours were characterized using von Frey filament, pinprick and thermal endpoints and then pharmacologically evaluated with known reference agents. RESULTS: Intraplantar NGF injection produced a dose-dependent increase in thermal sensitivity that lasted through 24 h post-injection and an immediate long-lasting (2 week) increase in mechanical sensitivity at the injection site, with no effects detected at secondary sites. NGF-induced mechanical sensitivity was pharmacologically characterized at 4 h and 1 week post-NGF injection. The nonsteroidal anti-inflammatory drugs (NSAIDs), celecoxib and diclofenac, were minimally effective against both thermal and mechanical endpoints. Gabapenitn and duloxetine were only moderately effective against thermal and mechanical hypersensitivity. Morphine was effective against thermal and mechanical endpoints at every time point examined. Treatment with the transient receptor potential vanilloid 1 (TRPV1) antagonist A-784168 partially attenuated NGF-induced thermal and mechanical sensitivity at all time points examined. CONCLUSIONS: The results reported here suggest that effects of NGF on thermal and mechanical sensitivity in rats are similar to those reported in human and are partially driven by TRPV1. The rat NGF model may serve as a potential translational model for exploring the effects of novel analgesic agents.

Assessing carrageenan-induced locomotor activity impairment in rats: comparison with evoked endpoint of acute inflammatory pain.

BACKGROUND: Most animal models currently used to evaluate antinociceptive efficacy of analgesics rely on the assessment of evoked pain behaviours as primary endpoints. METHODS: Here, we have developed and characterized the carrageenan-induced locomotor activity impairment (CLAIM) model to objectively assess non-evoked inflammatory pain behaviour in rats. In this model, 100 µL of 1% carrageenan was subcutaneously injected into the plantar aspect of the right hind paw and exploratory behaviour in the novel testing chamber was recorded using an automated locomotor activity system. RESULTS: Carrageenan-injected animals exhibited an exploratory behavioural deficit 2-7 h following injection compared to saline-injected animals. The severity of impairment was carrageenan dose related, and sensitive to the light intensity in the testing room. The effects of standard analgesics on CLAIM were examined 2 or 3 h following carrageenan injection. Diclofenac and ibuprofen, in a dose range exerting no effect on locomotor activity in naïve rats, exhibited dose-related reversal of CLAIM (ED(50) = 1.5 and 5.0 mg/kg, respectively), with comparable efficacy on carrageenan-induced thermal hyperalgesia (ED(50) = 2.0 and 6.0 mg/kg, respectively). Gabapentin and duloxetine produced no reversal of CLAIM, or attenuation of thermal hyperalgesia. Efficacy discrepancy was noted for morphine on thermal hyperalgesia and CLAIM. Additionally, amphetamine dose dependently reversed CLAIM, and similarly increased locomotor activity in normal animals. DISCUSSION AND CONCLUSION: The results presented here demonstrate that CLAIM provides an objective assessment of non-evoked pain behaviours for acute inflammatory pain. The pharmacological profile of standard analgesics supports that CLAIM model can be used to identify agents to treat acute inflammatory pain in the clinic.

Changes in exploratory behavior as a measure of chronic central pain following spinal cord injury.

Spinal cord injury (SCI) produces abnormal pain syndromes in patients that lead to changes in evoked and spontaneous behaviors. To test if a spontaneous component of pain-like behavior could be measured in a rodent model of chronic central pain (CCP), exploratory behavior (rearing events, rearing time, active time, rest time, distance traveled, and total activity) of adult male rats, subjected to sham surgery or spinal cord contusion injury treated with either vehicle (saline) or gabapentin (30 mg/kg, i.p.), was recorded. SCI was produced at spinal segment T10 using the NYU impactor device (10-g rod, 2.0-mm diameter, 12.5-mm drop height). Activity measures were collected on postsurgical days (PSD) 14, 28, and 60, and compared to presurgical activity. Sham control activity was not significantly different compared to presurgical activity in any measured parameter. SCI vehicle-treated rats demonstrated a significant decrease in total rearing time on PSD 14 and by PSD 28 significant differences in total activities where seen in all parameters measured. SCI gabapentin-treated rats did not display differences in total rearing time until PSD 28 and a significant difference in total activity of all measured parameters was not seen until PSD 60. No difference in hindlimb locomotor ability between SCI groups or sedation effects of gabapentin was found using open field BBB scores. We interpret the differences in exploratory behavior to reflect spontaneous behavioral changes due to CCP since (1) when locomotor ability was greatest, activity was lowest and (2) gabapentin attenuates the temporal decrease in activity. This study demonstrates that spontaneous as well as evoked behaviors may be used to evaluate CCP following SCI.

Involvement of metabotropic glutamate receptors in excitatory amino acid and GABA release following spinal cord injury in rat.

Spinal cord injury (SCI) leads to an increase in extracellular excitatory amino acid (EAA) concentrations resulting in glutamate receptor-mediated excitotoxic events. The glutamate receptors include ionotropic (iGluRs) and metabotropic (mGluR) receptors. Of the three groups of mGluRs, group-I activation can initiate intracellular pathways that lead to further transmitter release. Groups II and III mGluRs function mainly as autoreceptors to regulate neurotransmitter release. In an effort to examine the role of mGluRs in the increase in EAAs following SCI, we administered AIDA, a potent group-I mGluR antagonist immediately after injury. To determine subtype specific roles of the group-I mGluRs, we evaluated EAA release following LY 367385 (mGluR1 antagonist) and MPEP (mGluR5 antagonist) administration. To evaluate group-II and -III mGluRs we administered APDC (group-II agonist) and L-AP4 (group-III agonist) immediately following injury; additionally, we initiated treatment with CPPG (group-II/-III antagonist) and LY 341495 (group-II antagonist) 5 min prior to injury. Subjects were adult male Sprague-Dawley rats (225-250 g), impact injured at T10 with an NYU impactor (12.5 mm drop). Agents were injected into the epicenter of injury, amino acids where collected by microdialysis fibers inserted 0.5 mm caudal from the edge of the impact region and quantified by HPLC. Treatment with AIDA significantly decreased extracellular EAA and GABA concentrations. MPEP reduced EAA concentrations without affecting GABA. Combining LY 367385 and MPEP resulted in a decrease in EAA and GABA concentrations greater than either agent alone. L-AP4 decreased EAA levels, while treatment with LY 341495 increased EAA levels. These results suggest that mGluRs play an important role in EAA toxicity following SCI.

Strain and model differences in behavioral outcomes after spinal cord injury in rat.

Spinal cord injury (SCI) results in loss of function below the level of injury and the development of chronic central pain (CCP) syndromes. Since different strains may develop and express chronic pain behaviors differently, we evaluated behavioral outcomes (locomotor recovery and the development of mechanical and thermal allodynia) in three commonly used strains of rats (Long-Evans, Wistar, and Sprague-Dawley) using two models of SCI. The two models examined were contusion at T10 (NYU impactor, 12.5 mm height) and the T13 hemisection. Mechanical stimulation (von Frey filaments) revealed significantly lower baseline responses for Long-Evans rats and significantly higher baseline paw withdrawal latencies to thermal stimulation for Wistar rats compared to the other strains. Following contusion SCI, Long-Evans rats had the highest percentage of animals that developed mechanical allodynia (73%), while Sprague-Dawley rats had the highest percentages (75%) following hemisection SCI. Interestingly, the Sprague-Dawley rats had the highest percentage (87%) to develop thermal allodynia following contusion SCI, while 100% of both Long-Evans and Sprague Dawley rats developed thermal allodynia in the hemisection model. Locomotor recovery after SCI was similar for each model in that Long-Evans rats recovered slower and to a lesser extent than the other strains. In each model, Sprague-Dawley rats recovered faster and achieved greater function. Overall, the hemisection model produced a larger percentage of animals that developed CCP and had greater responses to mechanical stimulation. Thus, it appears that strain selection has a greater impact on locomotor recovery and model selection has a greater impact on the development of CCP following SCI. Furthermore, these results suggest that genetic factors may play a role in recovery following SCI.

Changes in metabotropic glutamate receptor expression following spinal cord injury.

Spinal cord injury (SCI) initiates biochemical events that lead to an increase in extracellular excitatory amino acid concentrations, resulting in glutamate receptor-mediated excitotoxic events. These receptors include the three groups of metabotropic glutamate receptors (mGluRs). Group I mGluR activation can initiate a number of intracellular pathways that increase neuronal excitability. Group II and III mGluRs may function as autoreceptors to modulate neurotransmission. Thus, all three groups may contribute to the mechanisms of central sensitization and chronic central pain. To begin evaluating mGluRs in SCI, we quantified the changes in mGluR expression after SCI in control (naive), sham, and impact injured adult male Sprague-Dawley rats (200-250 g). SCI was produced at spinal segment T10 with a New York University impactor (12.5-mm drop, 10-g rod of 2-mm diameter). Expression levels were determined by Western blot and immunohistochemistry analyses at the epicenter of injury, as well as segments rostral and caudal. The group I subtype mGluR1 was increased over control levels in segments rostral and caudal by postsurgical day (PSD) 7 and remained elevated through PSD 60. The group I subtype mGluR5 was unchanged in all segments rostral and caudal to the injury at every time point measured. Group II mGluRs were decreased compared to control levels from PSD 7 through PSD 60 in all segments. These results suggest that different subtypes of mGluRs have different spatial and temporal expression patterns following SCI. The expression changes in mGluRs parallel the development of mechanical allodynia and thermal hyperalgesia following SCI; therefore, understanding the expression of mGluRs after SCI may give insight into mechanisms underlying the development of chronic central pain.

Macrophage arginine metabolism to ornithine/urea or nitric oxide/citrulline: a life or death issue.

Macrophages can metabolize arginine to nitric oxide in quantities that inhibit pathogens or nearby host cells. They can instead metabolize arginine to ornithine (a precursor of polyamines and collagen) in quantities that stimulate pathogens or nearby host cells. Macrophages are essentially the only circulating cells that can make these life or death decisions with arginine. Macrophages expressing these destructive or constructive phenotypes have been termed M-1 or M-2 because they also stimulate TH1 or TH2 responses, respectively. Factors that influence whether a macrophage expresses the M-1 or M-2 phenotype and the real or potential impact on immune responses and other host processes are discussed.

AIDA reduces glutamate release and attenuates mechanical allodynia after spinal cord injury.

Spinal cord injury (SCI) leads to an increase in extracellular excitatory amino acid (EAA) concentrations, resulting in glutamate receptor-mediated excitotoxicity and central sensitization. To test contributions of group I metabotropic glutamate receptors (mGluRs) in SCI induced release of glutamate and in behavioral outcomes of central sensitization following injury, we administered 1-aminoindan-1,5-dicarboxylic acid (AIDA; 0.1 nmol intraspinally), a potent group I mGluR antagonist, to rats immediately after spinal cord contusion injury. EAAs were collected by microdialysis and quantified using HPLC. AIDA significantly decreased extracellular glutamate but not aspartate concentrations and significantly attenuated the development of mechanical but not thermal allodynia. These results suggest mGluRs play an important role in injury-induced EAA release and in central sensitization following SCI.

M-1/M-2 macrophages and the Th1/Th2 paradigm.

Evidence is provided that macrophages can make M-1 or M-2 responses. The concept of M-1/M-2 fomented from observations that macrophages from prototypical Th1 strains (C57BL/6, B10D2) are more easily activated to produce NO with either IFN-gamma or LPS than macrophages from Th2 strains (BALB/c, DBA/2). In marked contrast, LPS stimulates Th2, but not Th1, macrophages to increase arginine metabolism to ornithine. Thus, M-1/M-2 does not simply describe activated or unactivated macrophages, but cells expressing distinct metabolic programs. Because NO inhibits cell division, while ornithine can stimulate cell division (via polyamines), these results also indicate that M-1 and M-2 responses can influence inflammatory reactions in opposite ways. Macrophage TGF-beta1, which inhibits inducible NO synthase and stimulates arginase, appears to play an important role in regulating the balance between M-1 and M-2. M-1/M-2 phenotypes are independent of T or B lymphocytes because C57BL/6 and BALB/c NUDE or SCID macrophages also exhibit M-1/M-2. Indeed, M-1/M-2 proclivities are magnified in NUDE and SCID mice. Finally, C57BL/6 SCID macrophages cause CB6F1 lymphocytes to increase IFN-gamma production, while BALB/c SCID macrophages increase TGF-beta production. Together, the results indicate that M-1- or M-2-dominant macrophage responses can influence whether Th1/Th2 or other types of inflammatory responses occur.

Augmentation of an antitumor CTL response In vivo by inhibition of suppressor macrophage nitric oxide.

Evidence is provided that inhibition of macrophage NO production can augment in vivo CTL responses. Specifically, administration of NG-monomethyl-l -arginine (NGMMA) via osmotic pumps increases the tumor-specific CTL response against the P815 mastocytoma in the peritoneal cavity of preimmunized mice. Both the magnitude and duration of the CTL response were increased. That the augmented CTL response resulted from inhibition of the NO synthase pathway is supported by the finding that macrophage NO production from NGMMA-treated mice was reduced. Also, in vitro inhibition of NO production by peritoneal exudate cells from P815 tumor-challenged mice augmented the secondary CTL response observed. Cell proliferation was augmented by NGMMA in these cultures, suggesting that macrophage NO may suppress CTL by inhibiting clonal expansion. NO-mediated inhibition was observed in vivo in this experimental system, even though the CTL response is not suppressed, in that tumor rejection occurs. Therefore, the present results are consistent with the conclusion that macrophage NO-mediated inhibition of the CTL response is a side effect of activating macrophages rather than resulting from the action of a distinct subset of what have long been termed suppressor macrophages. Most important, the results indicate that NO-mediated suppressor macrophage activity can be an important CTL immunoregulatory element in vivo.

Evaluation of a rapid and inexpensive dipstick assay for the diagnosis of Plasmodium falciparum malaria.

Rapid, accurate and affordable methods are needed for the diagnosis of malaria. Reported here is an evaluation of a new immunochromatographic strip, the PATH Falciparum Malaria IC Strip, which is impregnated with an immobilized IgM monoclonal antibody that binds to the HRP-II antigen of Plasmodium falciparum. In contrast to other commercially available kits marketed for the rapid diagnosis of falciparum malaria, this kit should be affordable in the malaria-endemic world. Using microscopy and polymerase chain reaction (PCR)-based methods as reference standards, we compared two versions of the PATH test for the detection of P. falciparum infection in 200 febrile travellers. As determined by PCR and microscopy, 148 travellers had malaria, 50 of whom (33.8%) were infected with P. falciparum. Compared with PCR, the two versions of the PATH test had initial sensitivities of 90% and 88% and specificities of 97% and 96%, respectively, for the detection of falciparum malaria. When discrepant samples were retested blindly with a modified procedure (increased sample volume and longer washing step) the sensitivity and specificity of both kits improved to 96% and 99%, respectively. The two remaining false negatives occurred in samples with < 100 parasites per microliter of blood. The accuracy, simplicity and predicted low cost may make this test a useful diagnostic tool in malaria-endemic areas.

Increased susceptibility of male rats to kanamycin-induced cochleotoxicity.

Although clinical observations suggest that males are more susceptible than females to ototoxic drugs, controlled experimental studies investigating gender susceptibility have not been performed. Aminoglycosides initially attack the cochlea's outer hair cells (OHCs). We investigated the effects of the aminoglycoside, kanamycin, on electrophysiological function of OHCs in male and female rats. Animals were grouped by gender and treated with kanamycin (400 mg/kg/day kanamycin base, intramuscular injection) or equivolume normal saline. Administration was continued until distortion product otoacoustic emissions (DPOAEs) suggested a loss in OHC function in kanamycin-treated rats. Males treated with kanamycin showed changes in DPOAE thresholds and amplitudes as early as treatment day 10 which spread to all test frequencies by treatment day 13. In contrast, females treated with kanamycin did not show significant changes in thresholds or amplitudes until treatment day 22. The mechanism of increased male susceptibility to kanamycin cochleotoxicity has not been determined.

Antitumor response elicited by a superantigen-transmembrane sequence fusion protein anchored onto tumor cells.

Superantigens stimulate T cells bearing certain TCR beta-chain variable regions when bound to MHC II molecules. We investigated whether the superantigen toxic shock syndrome toxin-1 (TSST1) could induce an antitumor immune response when anchored onto MHC II-negative tumor cells. Our approach was to facilitate association of TSST1 with cell membranes by fusing its coding region to the transmembrane region (TM) sequence of the proto-oncogene c-erb-B-2. TSST1-TM was expressed in bacteria with an N-terminal histidine tag and purified using nickel-agarose affinity chromatography. Purified TSST1-TM added to cultures of several different MHC II-negative tumor cells spontaneously associated with cell membranes, as detected by flow cytometry. Because superantigens can direct cell-mediated cytotoxicity against MHC II-positive cells, a TM fusion protein lacking the TSST1 MHC II binding domain (TSST(88-194)-TM) was also constructed. Tumor cells precoated with TSST1-TM or TSST(88-194)-TM stimulated proliferation of human peripheral blood lymphocytes in vitro whereas uncoated tumor cells did not. Mice preimmunized with TSST1-TM- or TSST(88-194)-TM-coated tumor cells mounted a systemic response that resulted in significant antitumor immunity as measured by regression of a parental tumor challenge. TSST1-TM and TSST(88-194)-TM fusion proteins represent a useful new strategy for attaching superantigens or potentially other proteins onto tumor cell surfaces without genetic manipulation.

Comparison of the ParaSight-F test and the ICT Malaria Pf test with the polymerase chain reaction for the diagnosis of Plasmodium falciparum malaria in travellers.

Rapid and accurate methods are needed for the diagnosis of imported malaria. The ParaSight-F test and the ICT Malaria Pf test are commercially available kits marketed for the diagnosis of Plasmodium falciparum malaria. Both tests are antigen-capture assays based on the detection of P. falciparum histidine-rich protein 2 in peripheral blood. Using microscopy and a polymerase chain reaction (PCR)-based method as reference standards, we performed a 'blinded' comparison of these assays for the detection of P. falciparum infection in 200 febrile travellers returning from malaria-endemic areas. As determined by PCR and microscopy, 148 travellers had malaria and, of these patients, 54.7% (81/148) were infected with P. vivax only, 31.1% (46/148) with P. falciparum only, 9.5% (14/148) with P. ovale, 0.7% (1/148) with P. malariae, and 4.1% (6/148) had mixed infections. Compared to PCR, the ParaSight-F and ICT Malaria Pf tests had initial sensitivities of 94% and 90% and specificities of 95% and 97%, respectively, for the detection of P. falciparum malaria. When discrepant samples were retested with day 0 and day 1 bloods, the sensitivities improved to 96% and 94%, respectively. The 2 remaining false negative results with the Para-Sight-F test and 2 of the 3 false negative results with the ICT Malaria Pf test occurred in samples with < 100 parasites/microL. The performance of these kits was not significantly different (P = 0.75) and both are simple, rapid, and accurate tests for the detection of P. falciparum infection in the returned traveller.

Quantification of furosemide from serum and tissues using high-performance liquid chromatography.

Since pharmacokinetics may play a significant role in furosemide (FSM) developmental ototoxicity, we developed an assay for the extraction and quantification of FSM in tissue and fluid from neonatal and adult rats. Rats from post-natal day (PND) 10, 30 and 50, were given an intravenous dose of FSM (35 mg/kg). Blood and tissues were analyzed by HPLC. FSM in serum, perilymph and liver was elevated in PND ten rats as was the body burden of FSM. Renal concentrations were higher in older rats. Altered clearance of FSM in developing rats may result in higher concentrations in the cochlea and ototoxicity.

Differential regulation of macrophage arginine metabolism: a proposed role in wound healing.

Nitric oxide (NO) and ornithine, products of NO synthase or arginase, respectively, have opposing biological activities. The effect of mediators of leukocyte activation and inhibition on arginine metabolism of resident mouse peritoneal exudate cells (MPEC) was determined. Factors that increased basal NO synthase activity, interferon (IFN)-gamma and lipopolysaccharide (LPS), decreased arginase activity in intact cells. Transforming growth factor (TGF)-beta1 decreased IFN-gamma-stimulated NO synthase activity and produced a reciprocal increase in urea and ornithine release. TGF-beta1 had no effect on the activity of these enzymes in LPS-stimulated MPEC. Corticosterone (Cort, 100 ng/ml) decreased the basal activity of both enzymes. However, Cort inhibited NO synthase activity and increased ornithine release in MPEC exposed to IFN-gamma or LPS. The difference between arginase activity in intact cells vs. that of cell lysates suggested intracellular inhibition of arginase activity. Products of NO synthase, NO and citrulline, were shown to inhibit MPEC arginase activity under maximal assay conditions. Intracellular pH was not altered by exposure of MPEC to LPS, IFN-gamma, TGF-beta, and Cort. This reciprocal change in arginine metabolism is proposed to be an important component of wound healing. Expression of NO synthase creates a cytotoxic environment that may be important to the early phase of wound healing. As wound healing progresses, increased arginase activity produces an environment favorable for fibroblast replication and collagen production.

Effects of anesthesia on polyamine metabolism and water content in the rat brain.

Because variances have been noted in brain putrescine levels of anesthetized rats (control, SHAM-operated), we investigated the effects of several anesthetics on polyamine metabolism and water content in the adult rat brain. Short duration (5 min) anesthesia was studied in three groups: ketamine:xylazine [KX; 40 and 8 mg/kg, respectively, intraperitoneal injection (IP)], urethane (UR; 1.5 g/kg, IP), and isoflurane (IF, initially 3.5% in 100% O2, followed by a maintenance dose of 2.5% IF in 100% O2). Effects of IF at longer duration (30 min) were also studied because this paradigm is often used in our laboratory. Control rats received no anesthesia (NA). Following decapitation, tissue samples were obtained from 3 bilateral brain regions: parietal cortex, motor area (CPm); parietal cortex, somatosensory area (CPs); and the pyriform cortex (CPF). The polyamines, spermidine and spermine, and their precursor, putrescine, were quantified by HPLC-fluorometric detection and brain water content was determined by wet-to-dry weight measures. KX decreased putrescine (54%) and spermidine (20%) in the CPs, increased spermine (24%) in the CPF, and increased water content in all brain regions. UR decreased putrescine (51%) and slightly increased water content (0.7%) in the CPF. Short duration IF decreased putrescine and spermidine in all brain regions; decreased spermine in the CPm, and increased water content in the CPF (0.8%). In contrast, longer duration IF increased putrescine (181%) and spermidine (23%) in the CPm, with no change in water content. Anesthetics produce region-specific changes in putrescine, polyamines, and water content in the rat brain which could contribute to the experimental variability.

Insulin down-regulates the inducible nitric oxide synthase pathway: nitric oxide as cause and effect of diabetes?

Evidence in this paper indicates that insulin can down-regulate the inducible nitric oxide synthase (iNOS) pathway in vivo. The iNOS pathway is up-regulated in diabetes-prone rats and mice and is associated with an autoimmune process. However, the results presented here indicate that macrophage nitric oxide (NO) production and iNOS mRNA expression are also elevated in rats or mice made diabetic by streptozotocin injection in which there is no primary autoimmune component. Insulin administration reduces NO production in autoimmune-prone and streptozotocin-induced diabetic rodents. Finally, insulin decreases macrophage NO production in normal hosts. These results indicate that the autoimmune paradigm is inadequate to explain increased NO in diabetes. As a potential mechanism to explain insulin-mediated regulation of NO production, TGF-1 may be involved because 1) macrophages from diabetic mice produce less TGF-beta1 than macrophages from normal hosts; 2) the circulating TGF-beta1 level is lower in diabetic mice; and 3) insulin administration increases circulating TGF-beta1 in normal mice. Together, these results provide evidence that increased NO in diabetes is not only a cause but also an effect of beta-cell destruction and results in part from a heretofore unrecognized immunomodulatory activity of insulin.

Nitric oxide mediates early dysfunction of rat and mouse islets after transplantation.

Evidence presented in this paper indicates that nitric oxide (NO), generated by a nonspecific "wound"-type of inflammation, is an important mediator of the early dysfunction of transplanted islets in rodents. Although allogeneic islets stimulate NO production to a greater degree than syngeneic islets, the amounts of NO produced after either are significantly elevated above baseline. Inhibition of NO production by N(G)-monomethyl-L-arginine (NMA), markedly decreases the time needed to restore euglycemia after intraportal transplantation of syngeneic islets in diabetic rats. The dose of NMA used was not observably toxic, with no significant changes in blood pressure, hepatic artery blood flow, serum hepatic enzyme levels, or in weight compared with control animals. In rat recipients of intraportal syngeneic transplants, evidence that NO is produced at the site of implantation includes (1) an early and transient increase in posttransplant hepatic vein nitrate levels (pretransplant, 90 microM; 24 hr, 230 microM; 48 hr, 250 microM; 72 hr, 170 microM; and 96 hr, 140 microM), (2) concurrent appearance of inducible NO synthase mRNA in liver extracts, and (3) immunohistochemical localization of inducible NO synthase within the transplanted islets. Suppression of NO production or inhibition of NO activity is a potential strategy to increase the early function and engraftment transplanted islets in the clinical setting.