Geldanamycin accelerated peripheral nerve regeneration in comparison to FK-506 in vivo.

FK-506 accelerates nerve regeneration and improves functional recovery in vivo; its immunosuppressive properties, however, limit its clinical utility. Geldanamycin (GA), a non-immunosuppressive agent, shares a common binding target (heat shock protein 90) with FK-506 and may accelerate nerve regeneration through a similar mechanism. GA has been shown to augment neurite outgrowth in vitro but has not been tested in vivo. The current study investigated the effect of GA on the rate of axonal regeneration and functional recovery following peripheral nerve injury. In the first experiment, Thy1-GFP transgenic rats underwent serial transmuscular imaging to quantify the rate of axonal regeneration following saphenous nerve crush injury. In subsequent experiments, Lewis rats underwent tibial nerve crush or transection-and-repair injuries and were assessed for functional recovery by walking track analysis. All animals were randomized to receive daily administration of FK-506 (2mg/kg), GA (0.2mg/kg), or a control vehicle (dimethyl sulfoxide, 1 mL/kg) starting 3 days prior to injury. Both GA and FK-506 significantly increased the rate of axonal regeneration following crush injury in Thy1-GFP rats. In Lewis rats undergoing tibial nerve crush injury, earlier functional recovery occurred at day 5 and day 6 in animals treated with FK-506 and GA respectively, vs. day 13 for controls. Over a truncated 21-day timeframe, Lewis rats undergoing tibial nerve transection-and-repair injury and treated with FK-506 regained function at day 16, whereas those treated with GA or the control vehicle did not regain normal function. GA-treated animals, however, did exhibit significant functional improvement vs. controls. The current study demonstrated that GA accelerates axonal regeneration and enhances functional recovery in vivo. Its ability to increase the rate at which peripheral axons regenerate is comparable to that of FK-506. GA, however, did not match the performance of FK-506 in injury models where Wallerian degeneration (WD) is ongoing in the distal stump. This provides evidence that FK-506 accelerates axonal regeneration through two parallel mechanisms: the first being its well-established effect on neurons; the second is likely a newly described, as-yet poorly defined mechanism that affects WD. Finally, given the decrease in observed toxicity with GA administration, it might be a suitable non-immunosuppressive alternative to FK-506 for accelerating peripheral nerve regeneration in cases of clinical nerve injury.

End-to-side neurorrhaphy and lateral axonal sprouting in a long graft rat model.

OBJECTIVE/HYPOTHESIS: Controversy exists regarding collateral axonal sprouting across an end-to-side neurorrhaphy to provide functional motor reinnervation of a target organ without compromise of the donor nerve. Rat models may be limited in the study of end-to-side repair given potential contamination from the proximal nerve stump of the recipient distal nerve and the use of antagonistic muscle groups for donor and recipient. The current study attempts to address these issues by using a rat model in which an end-to-side coaptation is performed with a long graft interposed between the intact donor tibial nerve and the divided, distal contralateral tibial nerve. MATERIALS AND METHODS: The graft used in proximal end-to-side coaptation consisted of both sciatic nerves in a donor syngeneic animal. The distal repair to the contralateral tibial nerve was done immediately or in a delayed fashion to allow potential motor axons to transverse the graft before division of the recipient tibial nerve. RESULTS: After 24 weeks, axons were noted to transverse the entire distance of the graft and into the contralateral distal posterior tibial nerve. A significant increase in axonal numbers was observed in the immediate repairs compared with the delayed. No animal recovered functional motor ability on the contralateral side as assessed by walking tracks. CONCLUSIONS: These findings suggest the importance of immediate distal neurotrophic factors in encouraging nerve regeneration even in a long graft end-to-side repair. Our model is successful in demonstrating innervation through an end-to-side coaptation but questions its use given the lack of motor recovery.

A new artificial nerve graft containing rolled Schwann cell monolayers.

This study hypothesized that introducing high numbers of Schwann cells in monolayers via a novel rolled graft architecture would promote robust nerve regeneration. The objective was to place adherent Schwann cells in artificial nerve grafts and to assess regeneration through the Schwann cell-laden grafts compared with that through acellular grafts and autografts. Schwann cells were isolated from neonatal Fisher rats. Small intestinal submucosa (SIS) was harvested from adult Fisher rats, cut into 7 mm x 8 cm pieces, and pinned out. Schwann cells were plated onto the strips, allowed to reach confluence, and subsequently rolled into a laminar structure and implanted across a 7-mm gap in the rat sciatic nerve (n = 12). Control animals received SIS conduits without Schwann cells (n = 11) or autograft repair (n = 12). At 10.5 weeks, functional regeneration through the Schwann cell-laden grafts, measured by both sciatic function index and extensor postural thrust testing, exceeded that through the cell-free grafts and approached that achieved through autografts. These results highlight the role of Schwann cells in nerve regeneration. Regenerative results approaching autograft levels in the Schwann cell-laden group suggest that this methodology may ultimately be useful in clinical nerve repair.

Portal venous ultraviolet B-irradiated donor alloantigen prevents rejection in circumferential rat tracheal allografts.

BACKGROUND: Before tracheal transplantation can be considered as a method of reconstruction in patients with extensive circumferential tracheal defects, we must achieve a state of nontoxic, donor-specific tolerance so that the risks of such a transplant do not outweigh the benefits. OBJECTIVE: Our objective was to determine whether a single intraportal injection of modified donor alloantigen achieves donor-specific immunosuppression for major histocompatibility complex-mismatched rat tracheal allografts. STUDY DESIGN: Buffalo (recipient) rats were pretreated with either a single portal-vein administration of ultraviolet B (UVB)-irradiated donor splenocytes (n = 4) or an intraportal inoculation of nonirradiated donor splenocytes (n = 4). Major histocompatibility complex-mismatched Lewis (donor) tracheal allograft segments were then grafted into treatment groups 7 days after donor-cell pretreatment. Tracheal rejection was assessed by histologic analysis, mucosal cilia motility, and in vitro immunologic assessment. RESULTS: The UVB-treated group demonstrated no acute or chronic rejection as well as complete functional recovery. In vitro immunologic assessment demonstrated a donor-specific hyporesponsiveness and donor allospecificity. Untreated animals and those receiving nonirradiated donor splenocytes showed acute rejection of their tracheal allografts. CONCLUSION: Recipient pretreatment with intraportally administered UVB-irradiated donor splenocytes prevents rejection of circumferential rat tracheal allograft segments by inducing a donor-specific immune hyporesponsiveness.

Rat-strain differences in recovery following peripheral-nerve allotransplantation.

The rat model is commonly utilized in peripheral nerve research. Due to the short length of the rat limb and the animal's inherent neuroregenerative capacity, the timing of assessment of nerve regeneration is critical, and significant differences between groups can be lost if assessment is done too late. Additionally, the comparison of data from different rat-strain combinations has been questioned. This study better defines the time course of recovery after peripheral nerve grafting, and examines differences between Buffalo (BUF), Lewis (LEW), and ACI rats. Tibial-nerve isografts and allografts were performed and harvested at 6, 8, 10, or 14 weeks. Histomorphometry documented a statistically significant difference in the ACI/LEW and LEW/LEW combination at 10 weeks. No strain differences in graft rejection were noted. The optimal time to assess for histomorphometric differences in the ACI/LEW and LEW/LEW combination is at 10 weeks postoperatively.

FK506 rescues peripheral nerve allografts in acute rejection.

This study investigated the ability of the immunosuppressant FK506 to reverse nerve allograft rejection in progress. Eighty-four Buffalo rats received posterior tibial nerve grafts from either Lewis or Buffalo donor animals. Allografts were left untreated for either 7, 10, or 14 days before receiving daily subcutaneous FK506 injections (2 mg/kg). Time-matched control animals received either an isograft, an allograft with continuous FK506, or an allograft with no postoperative FK506 therapy. All animals underwent weekly evaluation of nerve function by walking track analysis. Experimental group animals were sacrificed either immediately prior to initiation of FK506 therapy (days 7, 10, or 14), after 2 weeks of immunosuppressive treatment, or 8 weeks postsurgery. Histomorphometric analysis, consisting of measurements of total number of nerve fibers, neural density, and percent of neural debris, demonstrated a statistically significant increase in regeneration in the isograft group relative to the untreated allograft group within 28 days of transplantation. Grafts harvested from animals receiving 2 weeks of FK506 after 7 or 10 days of rejection were histomorphometrically similar to time-matched isografts. By contrast, grafts from animals receiving 2 weeks of FK506 following 14 days without therapy resembled untreated allografts and demonstrated significant histomorphometric differences from isografts at the corresponding time point. Analysis of walking track data confirmed that relative to untreated allografts, functional recovery was hastened in animals receiving an isograft, or allograft treated with FK506. This study demonstrated that when started within 10 days of graft placement, FK506 could reverse nerve allograft rejection in rats evaluated following 2 weeks of treatment.

Pretreatment with portal venous ultraviolet B-irradiated donor alloantigen promotes donor-specific tolerance to rat nerve allografts.

OBJECTIVE: To determine if a single intraportal inoculation of ultraviolet B-irradiated (UVB) donor splenocytes can prevent nerve allograft rejection and confer donor-specific immunotolerance to rat nerve allograft segments. METHODS: Age-matched, class I and class II major histocompatibility complex (MHC) mismatched Buffalo (RT1b) rats were transplanted with a syngeneic nerve isograft, a Lewis (RT1l) nerve allograft, or a Brown-Norway (RT1n) rat nerve allograft segment. Control Buffalo rats in group I received a 3.0-cm Lewis (RT11) sciatic-posterior tibial interposition nerve allograft without pretreatment; group II Buffalo rats received a syngeneic Buffalo nerve isograft without pretreatment. Group III Buffalo recipients were inoculated with 2.5 x 107 UVB-irradiated Lewis donor splenocyte cells by portal venous administration 7 days before transplantation with a 3.0-cm sciatic-posterior tibial nerve allograft from a Lewis (RT11) or a third party Brown-Norway rat (RT1n) donor (group IV). Nerve graft regeneration was assessed with walking track analysis, nerve conduction studies, retrograde neural tracing, nerve graft histology, and morphometry. Recipient immune tolerance was assessed through in vitro immunological assessment. RESULTS: Pretreatment with UVB-irradiated donor splenocytes 7 days before transplantation prevented nerve allograft rejection. Pretreated animals receiving a nerve allograft recovered limb function, and demonstrated morphological, histological, and electrophysiologic parameters of nerve regeneration similar to that measured in rats receiving a nerve isograft. In vitro immunological assessment by mixed lymphocyte culture (MLC), cytotoxic T lymphocyte (CTL) assay, limiting dilution analysis (LDA) of helper (pTH) and cytotoxic (pCTL) precursor frequencies, and IL-2 production demonstrated a marked donor-specific suppression in allografted animals pretreated with intraportal UVB-irradiated donor splenocytes. These assessments correlated with indefinite acceptance of donor nerve allografts. CONCLUSIONS: A single pretreatment with a single intraportal dose of UVB-modified donor antigen specifically induces tolerance to peripheral nerve allografts in rats.

Development of the blood-nerve barrier in neonatal rats.

The blood-nerve barrier (BNB) is constituted by the perineurium and the endothelium of endoneurial microvessels. We investigated the age at which the vascular component of BNB function is established in the rat and the ultrastructural modifications accompanying changes in permeability. BNB permeability was assessed with injections of Evans blue albumin (EBA) and horseradish peroxidase (HRP) in rats of different ages. Sciatic nerve sections were studied using fluorescence and electron microscopy. Nerves from animals injected with EBA indicated that the BNB is not functional before 13 days of life but that its function is established by 16 days. These results were confirmed by electron microscope examination of nerve sections from animals injected with HRP, which showed clefts between the endothelial cells of endoneurial vessels in young rats. In rats over 18 days, these clefts were occluded by tight junctions, which prevented HRP from leaving the vessel lumen and conferred BNB function. Systematic morphometric analysis of nerves from different age groups allowed the establishment of baseline normal histologic neural development with age.

Effects of FKBP-12 ligands following tibial nerve injury in rats.

The neuroregenerative properties of FK506, an FKBP-12 ligand that inhibits calcineurin, and V-10,367, an FKBP-12 ligand that does not inhibit calcineurin, were evaluated in crush and transection models. Rats were randomly assigned to one of seven groups, including untreated controls and FK506- or V-10,367-treated experimental groups. Following crush or transection nerve injury, animals were assessed with walking tracks, and histomorphometry. FK506-treated animals demonstrated significant functional recovery 11 days following crush and 18 days following transection injury. In untreated and V-10,367 treated animals, nerves recovered 13 days following crush injury, but did not improve significantly prior to sacrifice at 28 days in animals sustaining a transection injury. No statistically significant differences in histomorphometric parameters were identified between any of the groups. The study confirms that FK506 accelerates recovery from tibial nerve injury.

Safe injection of cultured schwann cells into peripheral nerve allografts.

The effects of cultured host Schwann cells on axonal regeneration in peripheral nerve allografts were studied. Fischer rats served as recipient animals and Buffalo rats provided nerve allografts. Animals were randomized into 9 groups. Rats receiving tibial nerve isografts were left untreated (group I), or injected with isogeneic Fischer Schwann cells (group II) or placebo suspension (group III). Allografts obtained from Buffalo rats were left untreated (group IV), or received isogeneic Fischer Schwann cells (group V), 2 mg/kg Cyclosporin A and Fischer Schwann cells (group VI), 5 mg/kg Cyclosporin A (group VII), or 5 mg/kg Cyclosporin A with Schwann cells (group VIII). No Schwann cell tumors were identified 4 or 8 weeks postoperatively. Group IX animals, harvested 3 days postoperatively, demonstrated no evidence of injection injury. Schwann cells modestly improved axonal regeneration in both isografts and allografts and may have a clinical role in the treatment of peripheral nerve allografts.

Revascularization of peripheral nerve autografts and allografts.

The timing and mechanisms of peripheral nerve revascularization were investigated using a 2-cm sciatic nerve graft model in 58 rats. Epineurial perfusion was consistently established by 48 hours and endoneurial perfusion by 72 hours. The pattern of endoneurial perfusion was "all-or-none"--either all or none of the vessels in a fascicle exhibited blood flow. Conventional allografts exhibited similar revascularization dynamics and patterns. Capping the ends of the autograft with Silastic significantly delayed revascularization; no flow was observed at 4 days, and only a peripheral rim of perfused fascicular vessels was observed at 7 days. These patterns suggested that the primary method of revascularization in the conventional graft was longitudinal inosculation; no evidence of peripheral neovascularization or dependence on the graft bed as a source of revascularization was observed. The introduction of a major histocompatibility complex barrier between the grafted tissue and the recipient animal did not alter the timing or the mechanics of blood flow reestablishment.

Strain differences in peripheral-nerve regeneration in rats.

Currently, several strains of rats are used for studies of peripheral-nerve injury and repair. The purpose of this study was to determine if significant differences in regeneration between strains exist that might influence comparison of results and interpretation of scientific conclusions. One outbred (Sprague-Dawley) and four inbred stains (ACI, Wistar-Furth, Lewis, Brown-Norway) were studied. Animals were randomized to one of two experimental conditions, undergoing either posterior tibial nerve transection and repair, or Silastic conduit repair of the posterior tibial nerve (n=6/group). Endpoint evaluations at 6 and 13 weeks included histomorphometry and walking-track analysis. Evidence of excellent regeneration was noted in all rat strains undergoing primary repair. Generally, no statistically significant differences between strains were noted, regardless of endpoint evaluation used in the primary repair group. Nerve regeneration across the conduits was either poor or not present at 6 weeks, with no regeneration at all noted in any animals in the ACI and Brown-Norway groups, and regeneration in only one or two animals in the other strains. At 13 weeks, between three and five animals in each strain showed regeneration, but functional recovery was poor. Overall, few differences in peripheral-nerve recovery appear to exist between rat strains. It seems that uniform conclusions may be drawn regardless of strain used.

Cold preservation of nerve grafts decreases expression of ICAM-1 and class II MHC antigens.

Cold preservation has previously been shown to decrease the antigenicity of nerve allografts, while Schwann cells remain viable. The expression of intercellular adhesion molecule-1 (ICAM-1) and class II MHC antigens, both of which have been shown to play a major role in initiating graft rejection, was studied in fresh rat nerve, and after 2 and 7 weeks of cold preservation. Ten sciatic nerves harvested from Lewis rats were cut into three segments. One segment was processed immediately, while the other ones were preserved at 5 degrees C for 2 and 7 weeks, respectively, before processing. Immunostains using specific monoclonal antibodies and alkaline phosphatase development were performed on each sample. The relative level of expression of these antigens was compared using computer-assisted densitometry. Expression of ICAM-1 was significantly decreased at 7 weeks, as compared to fresh and 2-week groups, with no statistically significant difference between fresh and 2-week nerves. Expression of class II MHC was significantly decreased at 2 and 7 weeks, compared to fresh nerves, with no statistically significant difference between the preserved groups. The decrease in antigenicity of cold-preserved nerve allografts appears to be linked to a down-regulation of ICAM-1 and MHC class II expression.

Expression of 1-aminocyclopropane-1-carboxylate oxidase during leaf ontogeny in white clover.

We examined the expression of three distinct 1-aminocyclopropane-1-carboxylic acid oxidase genes during leaf ontogeny in white clover (Trifolium repens). Significant production of ethylene occurs at the apex, in newly initiated leaves, and in senescent leaf tissue. We used a combination of reverse transcriptase-polymerase chain reaction and 3'-rapid amplification of cDNA ends to identify three distinct DNA sequences designated TRACO1, TRACO2, and TRACO3, each with homology to 1-aminocyclopropane-1-carboxylic acid oxidase. Southern analysis confirmed that these sequences represent three distinct genes. Northern analysis revealed that TRACO1 is expressed specifically in the apex and TRACO2 is expressed in the apex and in developing and mature green leaves, with maximum expression in developing leaf tissue. The third gene, TRACO3, is expressed in senescent leaf tissue. Antibodies were raised to each gene product expressed in Escherichia coli, and western analysis showed that the TRACO1 antibody recognizes a protein of approximately 205 kD (as determined by gradient sodium dodecyl sulfate-polyacylamide gel electrophoresis) that is expressed preferentially in apical tissue. The TRACO2 antibody recognizes a protein of approximately 36.4 kD (as determined by gradient sodium dodecyl sulfate-polyacylamide gel electrophoresis) that is expressed in the apex and in developing and mature green leaves, with maximum expression in mature green tissue. No protein recognition by the TRACO3 antibody could be detected in senescent tissue or at any other stage of leaf development.

Regeneration across cold preserved peripheral nerve allografts.

The feasibility of peripheral nerve allograft pretreatment utilizing cold storage (5 degrees C in the University of Wisconsin Cold Storage Solution) or freeze-thawing to prevent rejection was investigated. Regeneration across cold-stored (3 or 5 weeks) or freeze-thawed (FT), 3.0-cm sciatic nerve allografts were compared to fresh auto- and allografts in an inbred rat model. At 16-week post-engraftment, only FT allografts appeared similar to autografts on gross inspection; FT grafts were neither shrunken nor adherent to the surrounding tissue as seen in the other allograft groups. Qualitatively, the pattern of regeneration in the graft segments of the fresh allograft and to a lesser extent of pretreated allografts was inferior to that of autografts as evidenced by a disruption in the perineurium, more extrafascicular axons, smaller and fewer myelinated axons, increased intrafascicular collagen deposition, and the persistence of perineurial cell compartmentation and perivascular infiltrates. Distal to these grafts, the regeneration became more homogenous between groups, although areas of ongoing Wallerian degeneration, new regeneration as well as compartmentation, were more prevalent in fresh and pretreated allografts. Although the number of myelinated fibres was equivalent to autografts, the fibre diameters, the number of large diameter fibres, and the G-ratio were significantly decreased in the allograft groups, which, in part, accounted for the significant decrease in conduction velocity in the 3-week stored and fresh allograft, and the slight decrease in the 5-week stored and FT allograft groups. There was a small return in the Sciatic Function Index towards normal, but no consistent differences between groups were found. Prolonged cold storage and freeze-thawing of nerve allografts resulted in regeneration that was better than fresh allografts, but inferior to autografts. With the concomitant use of host immunosuppression or other immunotherapies, these storage techniques can provide a means of transporting nerve allografts between medical centres and for converting urgent into elective procedures.

Peripheral nerve injection injury with antiemetic agents.

Antiemetics are widely used drugs, frequently administered to alleviate postoperative and postchemotherapeutic nausea and vomiting. While antiemetics do not induce peripheral neurotoxicity when administered systemically, it is not known whether peripheral nerve injury can occur as a result of inadvertent intraneural injection during intramuscular administration. The purpose of this study was to characterize the neurotoxic effect of three commonly used antiemetic agents (promethazine, dimenhydrinate, and prochlorperazine) as compared to saline in the rat sciatic nerve model. Intrafascicular and extrafascicular injection as well as direct application of the antiemetic drugs were performed. Nerves were harvested at 2 weeks postoperatively for histology and morphometry, with an additional sacrifice point at 8 weeks for the intrafascicular injection group. Injection injuries caused by antiemetic drugs differed depending on the agent injected and the location of injection. Extrafascicular injection and direct application caused no damage. Intrafascicular injection caused diffuse axonal injury in the promethazine and dimenhydrinate groups, while prochlorperazine caused only focal injury. Regeneration was prominent at 8 weeks in all intrafascicular injection groups in this rat model. Prochlorperazine thus appears to be less neurotoxic when injected intraneurally and should preferentially be used for intramuscular injections.

Regeneration through long nerve grafts in the swine model.

We investigated regeneration across a long nerve defect in the swine model to study extensive neural loss and long nerve gap. Most experiments have been conducted in the rodent model that, while an appropriate immunological model, only allows short nerve gaps to be studied. Twelve outbred swine received either an 8-cm ulnar nerve autograft or an allograft without immunosuppression. At 6 and 10 months, histomorphometry of the autografts demonstrated excellent nerve regeneration, while very poor regeneration was noted across the allografts. This confirmed that 8 cm are an adequate challenge independent of the spontaneous regeneration potential of axons seen in rodents. The swine ulnar nerve graft model causes minimal morbidity and will now be used with immunological manipulation of inbred animals.

Long-term observation of the effect of peripheral nerve injury in neonatal and young rats.

The purpose of this study was to observe functional recovery and motoneuron death after nerve transection-and-repair in neonatal versus young animals. One hundred nine Lewis rats underwent posterior tibial nerve transection-and-repair at 6 or 22 days of age. Fifty-two and fifty-seven nerves at the 6- and 22-day times were used for endpoint analysis at 1, 3, 10, and 14 months. These assessments included serial functional walking track analysis, electrophysiologic studies, muscle mass evaluation, motoneuron counts with retrograde horseradish peroxidase tracing, and histologic and morphometric nerve analysis. Walking track analysis and nerve conduction velocity indicated significantly poorer functional regeneration in the 6-day-old group than in the 22-day-old group. Muscle mass in the 6-day-old group did not recover as well as in the 22-day-old group. Motoneuron numbers stained with horseradish peroxidase were less in the 6-day-old group than in the 22-day-old group. In contrast, morphometric analysis did not reach significance. This study suggests that the same nerve injury sustained in a neonatal rat is less likely to demonstrate functional recovery than one sustained in a young rat.

End-to-side neurorraphy: a long-term study of neural regeneration in a rat model.

This study evaluated long-term reinnervation of an end-to-side neurorraphy and the resultant functional recovery in a rat model. The divided distal posterior tibial nerve was repaired to the side of an intact peroneal nerve. Control groups included a cut-and-repair of the posterior tibial nerve and an end-to-end repair of the peroneal nerve to the posterior tibial nerve. Evaluations included walking-track analysis, nerve conduction studies, muscle mass measurements, retrograde nerve tracing, and histologic evaluation. Walking tracks indicated poor recovery of posterior tibial nerve function in the experimental group. No significant difference in nerve conduction velocities was seen between the experimental and control groups. Gastrocnemius muscle mass measurements revealed no functional recovery in the experimental group. Similarly, retrograde nerve tracing revealed minimal motor neuron staining in the experimental group. However, some sensory staining was seen within the dorsal root ganglia of the end-to-side group. Histologic study revealed minimal myelinated axonal regeneration in the experimental group as compared with findings in the other groups. These results suggest that predominantly sensory regeneration occurs in an end-to-side neurorraphy at an end point of 6 months.

Nicotine replacement therapy.

OBJECTIVE: To review the literature on nicotine dependence, nicotine pharmacology, health consequences associated with the use of nicotine, and nicotine replacement therapies used to aid smokers who are nicotine dependent. DATA SOURCES: A review of articles, book bibliographies, and published studies identified by a search of the MEDLINE database from 1982 to 1996 on nicotine dependence, nicotine addiction, nicotine withdrawal, smoking, smoking cessation, smoking intervention, nicotine pharmacology, nicotine pharmacokinetics, nicotine pharmacodynamics, and nicotine replacement therapies. STUDY SELECTION AND DATA EXTRACTION: Inclusion criteria were published randomized, double-blind trials of at least 12 weeks' duration, meta-analyses, and panel consensus guidelines. DATA SYNTHESIS: Cigarette smoking and tobacco use have met the surgeon general's primary criteria as well as additional criteria for drug dependence. Drug dependence requires that the drug produce psychoactive effects. Nicotine has been identified as the cause of tobacco dependence. First, nicotine provides positive reinforcement by stimulating nicotinic receptors to promote high self-administration rates. Second, nicotine causes a negative reinforcement in the form of withdrawal symptoms when nicotine is withheld after chronic use. Nicotine replacement therapy reduces the severity of withdrawal symptoms in smokers abstaining from tobacco. Nicotine replacement therapy allows the smoker to focus on psychosocial aspects of tobacco abstinence while receiving relief from withdrawal symptoms. The long-term effectiveness and health benefits of nicotine replacement therapy coupled with nonpharmacologic approaches have been clearly established. Smoking cessation has received wide attention from the public and medical communities; it is complex and has several interwoven factors to be considered. The psychological, behavioral, and physical components have to be understood before designing a treatment plan. The most successful approaches to smoking cessation involve multicomponent, multisession behavioral treatment programs as a foundation coupled with pharmacologic intervention. Pharmacists can play a key role in initiating behavior change and ensuring the safe and proper use of nicotine replacement in order to produce the desired outcome. CONCLUSIONS: The optimum choice in nicotine replacement depends on the individual's needs and coping abilities. Individualized nicotine replacement coupled with nonpharmacologic interventions produces the highest rate of success for abstinence from nicotine.