Minocycline Before Aortic Occlusion Reduces Hindlimb Motor Impairment, Attenuates Spinal Cord Damage and Spinal Astrocytosis, and Preserve Neuronal Cytoarchitecture in the Rat.

OBJECTIVES: Spinal cord ischemia secondary to trauma or a vascular occlusive event is a threatening phenomenon. The neuroprotective properties of minocycline have been shown in several models of central nervous system diseases and after spinal cord ischemia; however, the benefit of using the drug requires additional confirmation in different animal models. Astrocytes are essential as regulators of neuronal functions and for providing nutrients. The authors hypothesized that astrocytes in the spinal cord may be an important target for minocycline action after ischemia and thus in the prevention of secondary spreading damage. DESIGN: A prospective, randomized animal study. SETTING: University research laboratory, single institution. PARTICIPANTS: Adult male Sprague Dawley rats, weighing between 400 and 450 g. INTERVENTIONS: A model of spinal cord ischemia in the rat was used for this study to determine whether a single, high-dose (10 mg/kg) of minocycline protects against damage to the neuronal cytoskeleton, both in the white and gray matter, and whether it reduces glial fibrillary acidic protein levels, which is an index for prevention of astrocyte activation during ischemia. Thirty minutes before thoracic aorta occlusion, minocycline was administered for 18 minutes using a 2 F Fogarty catheter. MEASUREMENTS AND MAIN RESULTS: Minocycline given prophylactically significantly mitigated severe hindlimb motor impairment and reduced glial fibrillary acidic protein plus astrocytosis in both the white and gray matter of the spinal cord, caudal to the occlusion. Neuronal histologic cytoarchitecture, which was severely and significantly compromised in control animals, was preserved in the minocycline-treated animals. CONCLUSIONS: This study's data imply that minocycline may attenuate reactive astrocytosis in response to injury with better neurologic outcome in a model of spinal cord ischemia in rats. The data suggest that future use of minocycline, clinically, might be advantageous in surgeries with a potential risk for paraplegia due to spinal cord ischemia.

Intrathecal Injection of miR-133b-3p or miR-143-3p Prevents the Development of Persistent Cold and Mechanical Allodynia Following a Peripheral Nerve Injury in Rats.

In DRG an increase in miR-133b-3p, miR-143-3p, and miR-1-3p correlates with the lack of development of neuropathic pain following a peripheral nerve injury. Using lentiviral (LV) vectors we found that a single injection of LV-miR-133b-3p or LV-miR-143-3p immediately after a peripheral nerve injury prevented the development of sustained mechanical and cold allodynia. Injection of LV-miR-133b-3p or LV-miR-143-3p by themselves or in combination, on day 3 post-injury produced a partial and transient reduction in mechanical allodynia and a sustained decrease in cold allodynia. Injection of LV-miR-1-3p has no effect. Co-injection of LV-miR-1a with miR-133b-3p or miR-143-3p on day 3 post-injury produced a sustained decrease in mechanical and cold allodynia. In DRG cultures, miR-133b-3p and miR-143-3p but not miR-1-3p, enhanced the depolarization-evoked cytoplasmic calcium increase. Using 3'UTR target clones containing a Gaussian luciferase reporter gene we found that with the 3'UTR-Scn2b, miR-133-3p and miR-143-3p reduced the expression while miR-1-3p enhanced the expression of the reporter gene. With the 3'UTR-TRPM8, miR-133-3p and miR-143-3p reduced the expression and miR-1-3p had no effect. With the 3'UTR-Piezo2, miR-133-3p increased the expression while miR-143-3p and miR-1-3p had no effect. LV-miR133b-3p, LV-miR-143-3p and LV-miR1a-3p reduced Scn2b-mRNA and Piezo2-mRNA. LV-miR133b-3p and LV-miR-143-3p reduced TRPM8-mRNA. LV-miR-133b-3p and LV-miR-143-3p prevent the development of chronic pain when injected immediately after the injury, but are only partially effective when injected at later times. LV-miR-1a-3p had no effect on pain, but complemented the actions of LV-miR-133b-3p or LV-miR-143-3p resulting in a sustained reversal of pain when co-injected 3 days following nerve injury.

Cannabinoid 1 receptor knockout mice display cold allodynia, but enhanced recovery from spared-nerve injury-induced mechanical hypersensitivity.

BACKGROUND: The function of the Cannabinoid 1 receptor (CB1R) in the development of neuropathic pain is not clear. Mounting evidence suggest that CB1R expression and activation may contribute to pain. Cannabinoid 1 receptor knockout mice (CB1R-/-) generated on a C57Bl/6 background exhibit hypoalgesia in the hotplate assay and formalin test. These findings suggest that Cannabinoid 1 receptor expression mediates the responses to at least some types of painful stimuli. By using this mouse line, we sought to determine if the lack of Cannabinoid 1 receptor unveils a general hypoalgesic phenotype, including protection against the development of neuropathic pain. The acetone test was used to measure cold sensitivity, the electronic von Frey was used to measure mechanical thresholds before and after spared-nerve injury, and analysis of footprint patterns was conducted to determine if motor function is differentially affected after nerve-injury in mice with varying levels of Cannabinoid 1 receptor. RESULTS: At baseline, CB1R-/- mice were hypersensitive in the acetone test, and this phenotype was maintained after spared-nerve injury. Using calcium imaging of lumbar dorsal root ganglion (DRG) cultures, a higher percentage of neurons isolated from CB1R-/- mice were menthol sensitive relative to DRG isolated from wild-type (CB1R+/+) mice. Baseline mechanical thresholds did not differ among genotypes, and mechanical hypersensitivity developed similarly in the first two weeks following spared-nerve injury (SNI). At two weeks post-SNI, CB1R-/- mice recovered significantly from mechanical hypersensitivity, while the CB1R+/+ mice did not. Heterozygous knockouts (CB1R+/-) transiently developed cold allodynia only after injury, but recovered mechanical thresholds to a similar extent as the CB1R-/- mice. Sciatic functional indices, which reflect overall nerve health, and alternation coefficients, which indicate uniformity of strides, were not significantly different among genotypes. CONCLUSION: Cold allodynia and significant recovery from spared-nerve injury-induced mechanical hypersensitivity are two novel phenotypes which characterize the global CB1R-/- mice. An increase in transient receptor potential channel of melastatin 8 channel function in DRG neurons may underlie the cold phenotype. Recovery of mechanical thresholds in the CB1R knockouts was independent of motor function. These results indicate that CB1R expression contributes to the development of persistent mechanical hypersensitivity, protects against the development of robust cold allodynia but is not involved in motor impairment following spared-nerve injury in mice.

Despite differences in cytosolic calcium regulation, lidocaine toxicity is similar in adult and neonatal rat dorsal root ganglia in vitro.

BACKGROUND: Neuraxial local anesthetics may have neurological complications thought to be due to neurotoxicity. A primary site of action of local anesthetics is the dorsal root ganglia (DRG) neuron. Physiologic differences have been noted between young and adult DRG neurons; hence, the authors examined whether there were any differences in lidocaine-induced changes in calcium and lidocaine toxicity in neonatal and adult rat DRG neurons. METHODS: DRG neurons were cultured from postnatal day 7 (P7) and adult rats. Lidocaine-induced changes in cytosolic calcium were examined with the calcium indicator Fluo-4. Cells were incubated with varying concentrations of lidocaine and examined for viability using calcein AM and ethidium homodimer-1 staining. Live imaging of caspase-3/7 activation was performed after incubation with lidocaine. RESULTS: The mean KCl-induced calcium transient was greater in P7 neurons (P < 0.05), and lidocaine significantly inhibited KCl-induced calcium responses in both ages (P < 0.05). Frequency distribution histograms of KCl-evoked calcium increases were more heterogeneous in P7 than in adult neurons. With lidocaine, KCl-induced calcium transients in both ages became more homogeneous but remained different between the groups. Interestingly, cell viability was decreased by lidocaine in a dose-dependent manner similarly in both ages. Lidocaine treatment also activated caspase-3/7 in a dose- and time-dependent manner similarly in both ages. CONCLUSIONS: Despite physiological differences in P7 and adult DRG neurons, lidocaine cytotoxicity is similar in P7 and adult DRG neurons in vitro. Differences in lidocaine- and KCl-evoked calcium responses suggest the similarity in lidocaine cytotoxicity involves other actions in addition to lidocaine-evoked effects on cytosolic calcium responses.

In vitro antiseptic effects on viability of neuronal and Schwann cells.

BACKGROUND AND OBJECTIVE: Chlorhexidine is recommended by several anesthesiology societies for antisepsis before regional anesthesia, but there is concern it may be neurotoxic. We evaluated the cytotoxicity of chlorhexidine and povidone-iodine in human neuronal and rat Schwann cells. METHODS: Human SH-SY5Y neuroblastoma and rat RSC96 Schwann cells were incubated with serial dilutions of 2% chlorhexidine gluconate and 10% povidone-iodine for 10 minutes, and viability was assessed with the MTT colorimetry assay and a fluorescent assay using calcein and ethidium. Cell morphology during antiseptic incubation was observed under light microscopy. To estimate the amount of antiseptic a needle carries through tissues, tritium radioactivity was measured in an animal injection model. RESULTS: Chlorhexidine at all tested concentrations significantly decreased viability compared with controls in both SH-SY5Y and RSC96 cells (P < 0.001). Povidone-iodine significantly decreased viability for both cells at concentrations of 0.2% or higher (P < 0.001). At the same dilutions of 1:200, 1:150, and 1:100, chlorhexidine was more cytotoxic than povidone-iodine for both cells (P< 0.001). During chlorhexidine treatment, both cell types became rounded and shriveled. Less dramatic changes were observed with povidone-iodine. In the injection model, 1.75% ± 1.29% of the maximum amount of radioactive contamination was carried through tissues. CONCLUSIONS: Chlorhexidine gluconate and povidone-iodine were cytotoxic to SH-SY5Y (neuronal) and RSC96 (Schwann) cells. Chlorhexidine was more potent than povidone-iodine at more dilute concentrations. However, the toxicity of the two was not different at concentrations used clinically. When using either of these agents for antisepsis before regional anesthesia, it is prudent to allow adequate drying time after application.

A novel liposomal bupivacaine formulation to produce ultralong-acting analgesia.

BACKGROUND: Currently available local anesthetics have relatively brief durations of action. An ultralong-acting local anesthetic would benefit patients with acute and chronic pain. The authors prepared and characterized a novel liposomal bupivacaine formulation using remote loading of bupivacaine along an ammonium sulfate gradient and assessed its efficacy in humans. METHODS: A large multivesicular liposomal bupivacaine formulation was prepared by subjecting small unilamellar vesicles to successive freeze-and-thaw cycles. Bupivacaine hydrochloride was then remotely loaded into the liposomes along an ammonium sulfate gradient ([(NH4)2SO4)]intraliposome/[(NH4)2SO4)]medium > 1000). The liposomes were then characterized for size distribution; drug-to-phospholipid ratio; in vitro release profile at 4 degree, 21 degree C, and 37 degree C; sterility; and pyrogenicity. Six subjects each received six intradermal injections in the lower back with 0.5 ml of 0.5, 1.0, and 2% liposomal bupivacaine; 0.5% standard bupivacaine; saline; and "empty" liposomes. Duration of analgesia was assessed using pinprick testing of the skin directly over the injection sites. Results were compared using the log-rank test. RESULTS: The mean large multivesicular vesicle size was 2439 +/- 544 nm, with a drug-to-phospholipid ratio of 1.8, fivefold greater than results previously reported. In vitro release was slowest at 4 degree C. The median duration of analgesia with 0.5% standard bupivacaine was 1 h. The median durations of analgesia after 0.5, 1.0, and 2.0% liposomal bupivacaine were 19, 38, and 48 h, respectively. Neither saline nor "empty" liposomes produced analgesia. CONCLUSIONS: This novel liposomal formulation had a favorable drug-to-phospholipid ratio and prolonged the duration of bupivacaine analgesia in a dose-dependent manner. If these results in healthy volunteers can be duplicated in the clinical setting, this formulation has the potential to significantly impact the management of pain.