Outcome in 12 Dogs with Chronic Radiographic Cranial Tibial Subluxation after Tibial Plateau Leveling Osteotomy (2019-2021).

Objective: The objective of this study was to examine outcomes in dogs with cruciate ligament rupture (CR) that had chronic radiographic cranial tibial subluxation at the time of osteotomy healing after tibial plateau leveling osteotomy (TPLO). Study Design. Retrospective case analysis of 12 dogs with prospective follow-up. Four of the 12 dogs were prospectively studied 12-24 months after surgery to assess long-term radiographic and clinical outcomes. Results: Three of the 4 dogs showed improvement in radiographic cranial tibial subluxation at long-term follow-up. In the other dog, minimally improved cranial tibial subluxation was associated with severe lameness. At long-term follow-up, gait analysis in 3 dogs with improved subluxation showed the symmetry of weight-bearing within 10% for peak vertical force, and no clinically lameness. Preoperative tibial plateau angle (TPA) and radiographic osteoarthritis in dogs with prospective follow-up and all dogs treated with TPLO surgery in the study period were not significantly different. Conclusion: Dogs with chronic radiographic cranial tibial subluxation are acceptable candidates for TPLO. Radiographic improvement in stifle reduction may take more than 10 weeks. The dog with long-term persistent subluxation also had a higher TPA over time, suggestive of ineffective surgical correction with TPLO and treatment failure.

Activated Mesenchymal Stromal Cell Therapy for Treatment of Multi-Drug Resistant Bacterial Infections in Dogs.

New and creative approaches are required to treat chronic infections caused by increasingly drug-resistant strains of bacteria. One strategy is the use of cellular therapy employing mesenchymal stromal cells (MSC) to kill bacteria directly and to also activate effective host immunity to infection. We demonstrated previously that activated MSC delivered systemically could be used effectively together with antibiotic therapy to clear chronic biofilm infections in rodent models. Therefore, we sought in the current studies to gain new insights into the antimicrobial properties of activated canine MSC and to evaluate their effectiveness as a novel cellular therapy for treatment of naturally-occurring drug resistant infections in dogs. These studies revealed that canine MSC produce and secrete antimicrobial peptides that synergize with most classes of common antibiotics to trigger rapid bactericidal activity. In addition, activated canine MSC migrated more efficiently to inflammatory stimuli, and secreted factors associated with wound healing and fibroblast proliferation and recruitment of activated neutrophils. Macrophages incubated with conditioned medium from activated MSC developed significantly enhanced bactericidal activity. Clinical studies in dogs with chronic multidrug resistant infections treated by repeated i.v. delivery of activated, allogeneic MSC demonstrated significant clinical benefit, including infection clearance and healing of infected tissues. Taken together, the results of these studies provide new insights into antimicrobial activity of canine MSC, and their potential clinical utility for management of chronic, drug-resistant infections.

Differentiating Bell's Palsy From Lyme-Related Facial Palsy.

BACKGROUND AND OBJECTIVES: To describe the etiology and clinical course of pediatric acute-onset unilateral peripheral facial palsy (FP), to define factors that distinguish Bell's palsy from Lyme-related FP (LRFP), and to determine if early corticosteroid use impacts facial strength recovery in Bell's palsy or LRFP. METHODS: Retrospective cohort study of children 1 to 18 years old who received clinical care within our pediatric clinical care network (Lyme-endemic region) between 2013 and 2018 for acute-onset unilateral peripheral FP. RESULTS: The study included 306 children; 82 (27%) had LRFP, 209 (68%) had Bell's palsy, and 15 (5%) had FP of different etiology. Most children with LRFP presented between June and November (93%), and compared with Bell's palsy, more often had a preceding systemic prodrome, including fever, malaise, headache, myalgias, and/or arthralgias (55% vs 6%, P < .001). Neuroimaging and lumbar puncture did not add diagnostic value in isolated FP. Of the 226 children with Bell's palsy or LRFP with documented follow-up, FP was resolved in all but 1. There was no association between ultimate parent/clinician assessment of recovery and early corticosteroid use. CONCLUSIONS: Bell's palsy and LRFP were common causes of pediatric FP in our Lyme endemic region. Systemic prodrome and calendar month may help distinguish LRFP from Bell's palsy at FP onset, guiding antibiotic use. Early corticosteroid use did not impact our measures of recovery, although subtle abnormalities may not have been appreciated, and time to recovery could not be assessed. Future prospective studies using standardized assessment tools at regular follow-up intervals are necessary.

Influenza-Associated Neurologic Complications in Hospitalized Children.

OBJECTIVES: To define the incidence and characteristics of influenza-associated neurologic complications in a cohort of children hospitalized at a tertiary care pediatric hospital with laboratory-confirmed influenza and to identify associated clinical, epidemiologic, and virologic factors. STUDY DESIGN: This was an historical cohort study of children aged 0.5-18.0 years old hospitalized between 2010 and 2017 with laboratory-confirmed influenza. Children with immune compromise or a positive test due to recent receipt of live virus vaccine or recently resolved illness were excluded. Influenza-associated neurologic complications were defined as new-onset neurologic signs/symptoms during acute influenza illness without another clear etiology. RESULTS: At least 1 influenza-associated neurologic complication was identified in 10.8% (95% CI 9.1-12.6%, n = 131 of 1217) of hospitalizations with laboratory-confirmed influenza. Seizures (n = 97) and encephalopathy (n = 44) were the most commonly identified influenza-associated neurologic complications, although an additional 20 hospitalizations had other influenza-associated neurologic complications. Hospitalizations with influenza-associated neurologic complications were similar in age and influenza type (A/B) to those without. Children with a pre-existing neurologic diagnosis (n = 326) had a greater proportion of influenza-associated neurologic complications compared with those without (22.7% vs 6.4%, P < .001). Presence of a pre-existing neurologic diagnosis (aOR 4.6, P < .001), lack of seasonal influenza vaccination (aOR 1.6, P = .020), and age ≤5 years (aOR 1.6, P = .017) were independently associated with influenza-associated neurologic complications. CONCLUSIONS: Influenza-associated neurologic complications are common in children hospitalized with influenza, particularly those with pre-existing neurologic diagnoses. A better understanding of the epidemiology and factors associated with influenza-associated neurologic complications will direct future investigation into potential neuropathologic mechanisms and mitigating strategies. Vaccination is recommended and may help prevent influenza-associated neurologic complications in children.

Experience of Chest Dysphoria and Masculinizing Chest Surgery in Transmasculine Youth.

OBJECTIVES: Transmasculine individuals, those assigned female sex at birth but who identify as masculine, have high rates of suicidal behavior and often suffer from chest dysphoria (discomfort and distress from unwanted breast development). Growing numbers of transmasculine youth are pursuing definitive treatment with masculinizing chest surgery (MCS), and adult studies reveal marked benefits of MCS, although little is known about the impact of chest dysphoria on transmasculine youth or the optimal timing of MCS. In this study, we aimed to explore youth experiences of chest dysphoria and the impact of MCS. METHODS: Transmasculine youth aged 13 to 21 were recruited from a pediatric hospital-based gender clinic. Participants completed a semistructured qualitative interview exploring the experience of chest dysphoria and thoughts about or experiences with MCS. Interview transcripts were coded by 3 investigators employing modified grounded theory, with the median interrater reliability at κ = 0.92. RESULTS: Subjects (N = 30) were a mean age of 17.5 years, and 47% had undergone MCS. Youth reported that chest dysphoria triggered strong negative emotions and suicidal ideation, caused a myriad of functional limitations, and was inadequately relieved by testosterone therapy alone. All post-MCS youth reported near or total resolution of chest dysphoria, lack of regret, and improved quality of life and functioning. CONCLUSIONS: We observed consensus that chest dysphoria is a major source of distress and can be functionally disabling to transmasculine youth. MCS performed during adolescence, including before age 18, can alleviate suffering and improve functioning. Additional research is needed to develop patient-reported outcome measures to assess the impact of chest dysphoria and MCS.

Findings on Colonoscopy after Diverticulitis: A Multicenter Review.

Diverticular disease is a common problem where patients with diverticulosis have a 1-4 per cent risk of acute diverticulitis. Current guidelines recommend a colonoscopy after.the resolution of acute diverticulitis. The aim of this study was to evaluate the yield of significant findings on colonoscopy after an episode of diverticulitis. This is a retrospective analysis of patients who underwent colonoscopy after an episode of diverticulitis between November 2005 and August 2017 at three major teaching hospitals. Advanced adenomas were defined as adenomas ≥1 cm, serrated adenomas, and tubulovillous or villous adenomas. A total of 584 patients (298 males; 51%) underwent colonoscopy for a history of diverticulitis after resolution of acute symptoms. Colonoscopy was complete in 488 patients (84%). Among these 488 patients, 446 had diverticular disease, 31 had advanced adenomas, and four had adenocarcinomas. Colonoscopies were incomplete in 96 patients (16%). Forty-six of those patients underwent surgery. The overall incidence of advanced adenomas and adenocarcinomas was 32 (5.4%) and nine (1.5%), respectively. In our study, the prevalence of advanced adenomas and adenocarcinomas was relatively high compared with the average risk individuals. Our findings support that patients after an episode of diverticulitis should continue to get a colonoscopy.

Expression of von Willebrand factor, pulmonary intravascular macrophages, and Toll-like receptors in lungs of septic foals.

Sepsis causes significant mortality in neonatal foals; however, there is little data describing the cellular and molecular pathways of lung inflammation in septic foals. This study was conducted to characterize lung inflammation in septic foals. Lung tissue sections from control (n = 6) and septic (n = 17) foals were compared using histology and immunohistology. Blinded pathologic scoring of hematoxylin and eosin stained samples revealed increased features of lung inflammation such as thickened alveolar septa and sequestered inflammatory cells in septic foals. Septic foal lungs showed increased expression of von Willebrand factor in blood vessels, demonstrating vascular inflammation. Use of MAC387 antibody to detect calprotectin as a reflection of mononuclear cell infiltration revealed a significant increase in their numbers in alveolar septa of lungs from septic foals compared to those from control foals. The mononuclear cells appeared to be mature macrophages and were located in the septal capillaries, suggesting they were pulmonary intravascular macrophages (PIMs). Finally, lungs from septic foals showed increased expression of Toll-like receptor 4 and 9 in mononuclear cells relative to the control. Taken together, this study is the first to show the expression of inflammatory molecules and an increase in PIMs in lungs from foals that died from sepsis.

Management of anastomotic leak: lessons learned from a large colon and rectal surgery training program.

BACKGROUND: Anastomotic leak is a dreaded surgical complication that can lead to significant morbidity and mortality. Despite its prevalence, there is no consensus on the management of anastomotic leak. This study aimed to review the management of anastomotic leak in the Division of Colon and Rectal Surgery at two institutions. METHODS: This is a retrospective review of all anastomotic leaks occurring after surgery in the Division of Colon and Rectal Surgery at two teaching institutions during 1997-2008. RESULTS: Altogether, 103 leaks occurred in 1,707 anastomoses (6 %), with a median time to diagnosis of 20 days (2-1,400 days). The 90-day mortality rate was 3 %. The majority of cases were managed nonoperatively (73 %), and the majority of leaks were from an extraperitoneal anastomosis (67 %). Success (i.e., radiographic demonstration of a healed leak, restored gastrointestinal continuity) occurred in 54 % of operatively managed leaks and 57 % of nonoperatively managed leaks (56 % overall). Operative management differed by leak location. In 91 % of patients with intraperitoneal leaks, the anastomosis was resected. In 76 % of patients with extraperitoneal leaks, diversion and drainage alone was performed without manipulating the anastomosis. Nonoperative management was successful for 57 % of extraperitoneal leaks and 58 % of intraperitoneal leaks. There was no significant difference in the success rates based on type of management (operative/nonoperative) for either extraperitoneal or intraperitoneal leaks. CONCLUSIONS: Anastomotic leak continues to result in patient morbidity and mortality. Its diverse presentation requires tailoring management to the patient. Nonoperative and operative treatments are viable options for intraperitoneal and extraperitoneal leaks based on patient presentation.

Intrathecal injection of adenosine 2A receptor agonists reversed neuropathic allodynia through protein kinase (PK)A/PKC signaling.

A single intrathecal dose of adenosine 2A receptor (A2AR) agonist was previously reported to produce a multi-week reversal of allodynia in a chronic constriction injury (CCI) model of neuropathic pain. We aimed to determine if this long-term reversal was induced by A2AR agonism versus more generalized across adenosine receptor subtypes, and begin to explore the intracellular signaling cascades involved. In addition, we sought to identify whether the enduring effect could be extended to other models of neuropathic pain. We tested an A1R and A2BR agonist in CCI and found the same long duration effect with A2BR but not A1R agonism. An A2AR agonist (ATL313) produced a significant long-duration reversal of mechanical allodynia induced by long established CCI (administered 6 weeks after surgery), spinal nerve ligation and sciatic inflammatory neuropathy. To determine if ATL313 had a direct effect on glia, ATL313 was coadministered with lipopolysaccharide to neonatal microglia and astrocytes in vitro. ATL313 significantly attenuated TNFα production in both microglia and astrocytes but had no effect on LPS induced IL-10. Protein kinase C significantly reversed the ATL313 effects on TNFα in vitro in microglia and astrocytes, while a protein kinase A inhibitor only effected microglia. Both intrathecal PKA and PKC inhibitors significantly reversed the effect of the A2AR agonist on neuropathic allodynia. Therefore, A2AR agonists administered IT remain an exciting novel target for the treatment of neuropathic pain.

Prior exposure to glucocorticoids potentiates lipopolysaccharide induced mechanical allodynia and spinal neuroinflammation.

While stress and stress-induced glucocorticoids are classically considered immunosuppressive, they can also enhance proinflammatory responses to subsequent challenges. Corticosterone (CORT) primes rat immune cells, exacerbating pro-inflammatory responses to subsequent immune challenges. Stress can also sensitize pain. One possibility is that stress primes spinal immune cells, predominantly glia, which are key mediators in pain enhancement through their release of proinflammatory cytokines. Therefore, we aimed to identify whether prior CORT sensitizes spinal cord glia such that a potentiated pro-inflammatory response occurs to later intrathecal (IT) lipopolysaccharide (LPS), thereby enhancing pain. Rats received subcutaneous CORT/vehicle 24 h before IT LPS/vehicle. Hind paw pain thresholds were measured before CORT/vehicle, before and up to 48 h after IT LPS/vehicle. In separate rats treated as above, lumbar spinal cord tissue was collected and processed for proinflammatory mediators. CORT alone had no effect on pain responses, nor on any pro-inflammatory cytokines measured. LPS induced allodynia (decreased pain threshold) lasting <4 h and elevated spinal IL-1ß and IL-6 protein. Prior CORT potentiated allodynia, lasting >24 h following LPS and potentiated spinal IL-1 and IL-6 protein. Coadministration of IL-1 receptor antagonist with LPS IT completely blocked the allodynia irrespective of whether the system was primed by CORT or not. At 24 h, TLR2, TLR4, MD2, and CD14 mRNAs were significantly elevated within the spinal cord in the CORT+LPS group compared to all other groups. Prior CORT before a direct spinal immune challenge is able to potentiate pain responses and pro-inflammatory cytokine production.

Caudal granular insular cortex is sufficient and necessary for the long-term maintenance of allodynic behavior in the rat attributable to mononeuropathy.

Mechanical allodynia, the perception of innocuous tactile stimulation as painful, is a severe symptom of chronic pain often produced by damage to peripheral nerves. Allodynia affects millions of people and remains highly resistant to classic analgesics and therapies. Neural mechanisms for the development and maintenance of allodynia have been investigated in the spinal cord, brainstem, thalamus, and forebrain, but manipulations of these regions rarely produce lasting effects. We found that long-term alleviation of allodynic manifestations is produced by discreetly lesioning a newly discovered somatosensory representation in caudal granular insular cortex (CGIC) in the rat, either before or after a chronic constriction injury of the sciatic nerve. However, CGIC lesions alone have no effect on normal mechanical stimulus thresholds. In addition, using electrophysiological techniques, we reveal a corticospinal loop that could be the anatomical source of the influence of CGIC on allodynia.

Pain intensity and duration can be enhanced by prior challenge: initial evidence suggestive of a role of microglial priming.

UNLABELLED: Activation of spinal microglia and consequent release of proinflammatory mediators facilitate pain. Under certain conditions, responses of activated microglia can become enhanced. Enhanced microglial production of proinflammatory products may result from priming (sensitization), similar to macrophage priming. We hypothesized that if spinal microglia were primed by an initial inflammatory challenge, subsequent challenges may create enhanced pain. Here, we used a "two-hit" paradigm using 2 successive challenges, which affect overlapping populations of spinal microglia, presented 2 weeks apart. Mechanical allodynia and/or activation of spinal glia were assessed. Initially, laparotomy preceded systemic lipopolysaccharide (LPS). Prior laparotomy caused prolonged microglial (not astrocyte) activation plus enhanced LPS-induced allodynia. In this "two-hit" paradigm, minocycline, a microglial activation inhibitor, significantly reduced later exaggerated pain induced by prior surgery when minocycline was administered intrathecally for 5 days starting either at the time of surgery or 5 days before LPS administration. To test generality of the priming effect, subcutaneous formalin preceded intrathecal HIV-1 gp120, which activates spinal microglia and causes robust allodynia. Prior formalin enhanced intrathecal gp120-induced allodynia, suggesting that microglial priming is not limited to laparotomy and again supporting a spinal site of action. Therefore, spinal microglial priming may increase vulnerability to pain enhancement. PERSPECTIVE: Spinal microglia may become "primed" (sensitized) following their activation by disparate forms of peripheral trauma/inflammation. As a result, such primed microglia may overrespond to subsequent challenges, thereby enhancing pain intensity and duration.

Intrathecal injection of an alpha seven nicotinic acetylcholine receptor agonist attenuates gp120-induced mechanical allodynia and spinal pro-inflammatory cytokine profiles in rats.

Nicotinic acetylcholine receptors (nAchRs) are not only key receptors in the autonomic nervous system, but also are present on immune cells. The alpha seven subunit of nAchR (alpha7nAchR) suppresses pro-inflammation in peripheral monocytes by decreasing pro-inflammatory cytokine production. In spinal cord, alpha7nAchRs are found on microglia, which are known to induce and maintain pain. We predicted that alpha7nAchR agonists might attenuate intrathecal HIV-1 gp120-induced, pro-inflammatory cytokine- and microglia-dependent mechanical allodynia. Choline, a precursor for acetylcholine and selective agonist for alpha7nAchR, was administered intrathecally either with, or 30 min after, intrathecal gp120. Choline significantly blocked and reversed gp120-induced mechanical allodynia for at least 4 h after drug administration. In addition, intrathecal choline, delivered either with or 30 min after gp120, reduced gp120-induced IL-1beta protein and pro-inflammatory cytokine mRNAs within the lumbar spinal cord. A second alpha7nAchR agonist, GTS-21, also significantly reversed gp120-induced mechanical allodynia and lumbar spinal cord levels of pro-inflammatory cytokine mRNAs and IL-1beta protein. A role of microglia is suggested by the observation that intrathecal choline suppressed the gp120-induced expression of, cd11b, a macrophage/microglial activation marker. Taken together, the data support that alpha7nAchR may be a novel target for treating pain where microglia maintain the pro-inflammatory state within the spinal cord.

Release of plasmid DNA-encoding IL-10 from PLGA microparticles facilitates long-term reversal of neuropathic pain following a single intrathecal administration.

PURPOSE: Interleukin-10 (IL-10) is an anti-inflammatory molecule that has achieved interest as a therapeutic for neuropathic pain. In this work, the potential of plasmid DNA-encoding IL-10 (pDNA-IL-10) slowly released from biodegradable microparticles to provide long-term pain relief in an animal model of neuropathic pain was investigated. METHODS: PLGA microparticles encapsulating pDNA-IL-10 were developed and assessed both in vitro and in vivo. RESULTS: In vitro, pDNA containing microparticles activated macrophages, enhanced the production of nitric oxide, and increased the production of IL-10 protein relative to levels achieved with unencapsulated pDNA-IL-10. In vivo, intrathecally administered microparticles embedded in meningeal tissue, induced phagocytic cell recruitment to the cerebrospinal fluid, and relieved neuropathic pain for greater than 74 days following a single intrathecal administration, a feat not achieved with unencapsulated pDNA. Therapeutic effects of microparticle-delivered pDNA-IL-10 were blocked in the presence of IL-10-neutralizing antibody, and elevated levels of plasmid-derived IL-10 were detected in tissues for a prolonged time period post-injection (>28 days), demonstrating that therapeutic effects are dependent on IL-10 protein production. CONCLUSIONS: These studies demonstrate that microparticle encapsulation significantly enhances the potency of intrathecally administered pDNA, which may be extended to treat other disorders that require intrathecal gene therapy.

PEGylation of interleukin-10 for the mitigation of enhanced pain states.

The anti-inflammatory cytokine interleukin-10 (IL-10) shows promise for the treatment of neuropathic pain, but for IL-10 to be clinically useful as a short-term therapeutic its duration needs to be improved. In this study, IL-10 was covalently modified with polyethylene glycol (PEG) with the goal of stabilizing and increasing protein levels in the CSF to improve the efficacy of IL-10 for treating neuropathic pain. Two different PEGylation methods were explored in vitro to identify suitable PEGylated IL-10 products for subsequent in vivo testing. PEGylation of IL-10 by acylation yielded a highly PEGylated product with a 35-fold in vitro biological activity reduction. PEGylation of IL-10 by reductive amination yielded products with a minimal number of PEG molecules attached and in vitro biological activity reductions of approximately 3-fold. In vivo collections of cerebrospinal fluid after intrathecal administration demonstrated that 20 kDa PEG attachment to IL-10 increased the concentration of IL-10 in the cerebrospinal fluid over time. Relative to unmodified IL-10, the 20 kDa PEG-IL-10 product exhibited an increased therapeutic duration and magnitude in an animal model of neuropathic pain. This suggests that PEGylation is a viable strategy for the short-term treatment or, in conjunction with other approaches, the long-term treatment of enhanced pain states.

Enduring reversal of neuropathic pain by a single intrathecal injection of adenosine 2A receptor agonists: a novel therapy for neuropathic pain.

Previous studies of peripheral immune cells have documented that activation of adenosine 2A receptors (A(2A)Rs) decrease proinflammatory cytokine release and increase release of the potent anti-inflammatory cytokine, interleukin-10 (IL-10). Given the growing literature supporting that glial proinflammatory cytokines importantly contribute to neuropathic pain and that IL-10 can suppress such pain, we evaluated the effects of intrathecally administered A(2A)R agonists on neuropathic pain using the chronic constriction injury (CCI) model. A single intrathecal injection of the A(2A)R agonists 4-(3-(6-amino-9-(5-cyclopropylcarbamoyl-3,4-dihydroxytetrahydrofuran-2-yl)-9H-purin-2-yl)prop-2-ynyl)piperidine-1-carboxylic acid methyl ester (ATL313) or 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamido adenosine HCl (CGS21680), 10-14 d after CCI versus sham surgery, produced a long-duration reversal of mechanical allodynia and thermal hyperalgesia for at least 4 weeks. Neither drug altered the nociceptive responses of sham-operated controls. An A(2A)R antagonist [ZM241385 (4-(2-[7-amino-2-(2-furyl)(1,2,4)triazolo(2,3-a)(1,3,5)triazin-5-ylamino]ethyl)phenol)] coadministered intrathecally with ATL313 abolished the action of ATL313 in rats with neuropathy-induced allodynia but had no effect on allodynia in the absence of the A(2A)R agonist. ATL313 attenuated CCI-induced upregulation of spinal cord activation markers for microglia and astrocytes in the L4-L6 spinal cord segments both 1 and 4 weeks after a single intrathecal ATL313 administration. Neutralizing IL-10 antibodies administered intrathecally transiently abolished the effect of ATL313 on neuropathic pain. In addition, IL-10 mRNA was significantly elevated in the CSF cells collected from the lumbar region. Activation of A(2A)Rs after intrathecal administration may be a novel, therapeutic approach for the treatment of neuropathic pain by increasing IL-10 in the immunocompetent cells of the CNS.

A peptide antagonist of the TLR4-MD2 interaction.

Toll-like receptors are an integral part of innate immunity in the central nervous system (CNS); they orchestrate a robust defense in response to both exogenous and endogenous danger signals. Recently, toll-like receptor 4 (TLR4) has emerged as a therapeutic target for the treatment of CNS-related diseases such as sepsis and chronic pain. We herein report a chemical biology approach by using a rationally designed peptide inhibitor to disrupt the TLR4-MD2 association, thereby blocking TLR4 signaling.

PEGylation of brain-derived neurotrophic factor for preserved biological activity and enhanced spinal cord distribution.

Brain-derived neurotrophic factor (BDNF) was covalently attached to polyethylene glycol (PEG) in order to enhance delivery to the spinal cord via the cerebrospinal fluid (intrathecal administration). By varying reaction conditions, mixtures of BDNF covalently attached to one (primary), two (secondary), three (tertiary), or more (higher order) PEG molecules were produced. The biological activity of each resulting conjugate mixture was assessed with the goal of identifying a relationship between the number of PEG molecules attached to BDNF and biological activity. A high degree of in vitro biological activity was maintained in mixtures enriched in primary and secondary conjugate products, while a substantial reduction in biological activity was observed in mixtures with tertiary and higher order conjugates. When a biologically active mixture of PEG-BDNF was administered intrathecally, it displayed a significantly improved half-life in the cerebrospinal fluid and an enhanced penetration into spinal cord tissue relative to native BDNF. Results from these studies suggest a PEGylation strategy that preserves the biological activity of the protein while also improving the half-life of the protein in vivo. Furthermore, PEGylation may be a promising approach for enhancing intrathecal delivery of therapeutic proteins with potential for treating disease and injury in the spinal cord.

Non-stereoselective reversal of neuropathic pain by naloxone and naltrexone: involvement of toll-like receptor 4 (TLR4).

Although activated spinal cord glia contribute importantly to neuropathic pain, how nerve injury activates glia remains controversial. It has recently been proposed, on the basis of genetic approaches, that toll-like receptor 4 (TLR4) may be a key receptor for initiating microglial activation following L5 spinal nerve injury. The present studies extend this idea pharmacologically by showing that TLR4 is key for maintaining neuropathic pain following sciatic nerve chronic constriction injury (CCI). Established neuropathic pain was reversed by intrathecally delivered TLR4 receptor antagonists derived from lipopolysaccharide. Additionally, (+)-naltrexone, (+)-naloxone, and (-)-naloxone, which we show here to be TLR4 antagonists in vitro on both stably transfected HEK293-TLR4 and microglial cell lines, suppressed neuropathic pain with complete reversal upon chronic infusion. Immunohistochemical analyses of spinal cords following chronic infusion revealed suppression of CCI-induced microglial activation by (+)-naloxone and (-)-naloxone, paralleling reversal of neuropathic pain. Together, these CCI data support the conclusion that neuron-to-glia signaling through TLR4 is important not only for initiating neuropathic pain, as suggested previously, but also for maintaining established neuropathic pain. Furthermore, these studies suggest that the novel TLR4 antagonists (+)-naloxone and (-)-naloxone can each fully reverse established neuropathic pain upon multi-day administration. This finding with (+)-naloxone is of potential clinical relevance. This is because (+)-naloxone is an antagonist that is inactive at the (-)-opioid selective receptors on neurons that produce analgesia. Thus, these data suggest that (+)-opioid antagonists such as (+)-naloxone may be useful clinically to suppress glial activation, yet (-)-opioid agonists suppress pain.