The formalin model is widely used for evaluating the effects of analgesic compounds in laboratory animals. Injection of formalin into the hind paw induces a biphasic pain response; the first phase is thought to result from direct activation of primary afferent sensory neurons, whereas the second phase has been proposed to reflect the combined effects of afferent input and central sensitization in the dorsal horn. Here we show that formalin excites sensory neurons by directly activating TRPA1, a cation channel that plays an important role in inflammatory pain. Formalin induced robust calcium influx in cells expressing cloned or native TRPA1 channels, and these responses were attenuated by a previously undescribed TRPA1-selective antagonist. Moreover, sensory neurons from TRPA1-deficient mice lacked formalin sensitivity. At the behavioral level, pharmacologic blockade or genetic ablation of TRPA1 produced marked attenuation of the characteristic flinching, licking, and lifting responses resulting from intraplantar injection of formalin. Our results show that TRPA1 is the principal site of formalin's pain-producing action in vivo, and that activation of this excitatory channel underlies the physiological and behavioral responses associated with this model of pain hypersensitivity.analgesia ͉ inflammation ͉ trp channel ͉ formaldehyde T he formalin model was developed Ͼ30 years ago to assess pain and evaluate analgesic drugs in laboratory animals (1). In this test, a dilute (0.5-5%) formalin solution (in which formaldehyde is the active ingredient) is injected into the paw of a rodent, and pain-related behaviors are assessed over two temporally distinct phases, including an initial robust phase in which paw lifting, licking, and f linching are scored during the first 10 min, followed by a transient decline in these behaviors and a subsequent second phase of behavior lasting 30 -60 min (2, 3).Compounds that typically affect the first phase (Phase I) include local anesthetics, such as lidocaine (4). The second phase (Phase II) is proposed to result from activity-dependent sensitization of CNS neurons within the dorsal horn (3, 5, 6). Many analgesics, including intrathecal nonsteroidal antiinflammatory drugs (7), NMDA antagonists (8, 9), morphine (1, 10), and gabapentin (11, 12), inhibit only Phase II responses, but not Phase I.The formalin test has several advantages over other models, in that spontaneous pain-related responses can be observed in a freely moving unrestrained animal. Once injected, no additional stimulus is required to evoke nocifensive behaviors, and behaviors can be scored over a prolonged period such that the precise onset and duration of analgesics can be assessed (1). However, despite the utility and widespread use of the formalin model in pain research, the mechanism by which formalin triggers C-fiber activation remains unknown (13) and is often attributed to tissue injury (1,3,9).In this study, we show that formalin activates primary afferent sensory neurons through a specific and direct action o...

TRPA1 is a nonselective cation channel expressed by nociceptors. Although it is widely accepted that TRPA1 serves as a broad irritancy receptor for a variety of reactive chemicals, its role in cold sensation remains controversial. Here, we demonstrate that mild cooling markedly increases agonist-evoked rat TRPA1 currents. In the absence of an agonist, even noxious cold only increases current amplitude slightly. These results suggest that TRPA1 is a key mediator of cold hypersensitivity in pathological conditions in which reactive oxygen species and proinflammatory activators of the channel are present, but likely plays a comparatively minor role in acute cold sensation. Supporting this, cold hypersensitivity can be induced in wild-type but not Trpa1 Ϫ/Ϫ mice by subcutaneous administration of a TRPA1 agonist. Furthermore, the selective TRPA1 antagonist HC-030031 [2-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydro-7H-purin-7-yl)-N-(4-isopropylphenyl)acetamide] reduces cold hypersensitivity in rodent models of inflammatory and neuropathic pain.

Pompe disease (glycogen storage disease II) is caused by mutations in the acid α-glucosidase gene. The most common form is rapidly progressive with glycogen storage, particularly in muscle, that leads to profound weakness, cardiac failure, and death by the age of two years. Although usually considered a muscle disease, glycogen storage also occurs in the CNS. We evaluated the progression of neuropathological and behavioral abnormalities in a Pompe disease mouse model (6neo/6neo) that displays many features of the human disease. Homozygous mutant mice store excess glycogen within large neurons of hindbrain, spinal cord, and sensory ganglia by the age of one month; accumulations then spread progressively within many CNS cell types. “Silver degeneration” and Fluoro-Jade C stains revealed severe degeneration in axon terminals of primary sensory neurons at three to nine months. These abnormalities were accompanied by progressive behavioral impairment on rotorod, wire hanging and foot fault tests. The extensive neuropathological alterations in this model suggest that therapy of skeletal and cardiac muscle disorders by systemic enzyme replacement therapy may not be sufficient to reverse functional deficits due to CNS glycogen storage, particularly early-onset, rapidly progressive disease. A better understanding of the basis for clinical manifestations is needed to correlate CNS pathology with Pompe disease manifestations.

Niemann-Pick type A disease is a lysosomal storage disorder caused by a deficiency in acid sphingomyelinase (ASM) activity. Previously we showed that storage pathology in the ASM knockout (ASMKO) mouse brain can be corrected by adeno-associated virus serotype 2 (AAV2)-mediated gene transfer. The present experiment compared the relative therapeutic efficacy of different recombinant AAV serotype vectors (1, 2, 5, 7, and 8) using histological, biochemical, and behavioral endpoints. In addition, we evaluated the use of the deep cerebellar nuclei (DCN) as a site for injection to facilitate global distribution of the viral vector and enzyme. Seven-week-old ASM knockout mice were injected within the DCN with different AAV serotype vectors encoding human ASM (hASM) and then killed at either 14 or 20 weeks of age. Results showed that AAV1 was superior to serotypes 2, 5, 7, and 8 in its relative ability to express hASM, alleviate storage accumulation, and correct behavioral deficits. Expression of hASM was found not only within the DCN, but also throughout the cerebellum, brainstem, midbrain, and spinal cord. This finding demonstrates that targeting the DCN is an effective approach for achieving widespread enzyme distribution throughout the CNS. Our results support the continued development of AAV based vectors for gene therapy of the CNS manifestations in Niemann-Pick type A disease.axonal transport ͉ deep cerebellar nuclei ͉ gene therapy ͉ lysosomal storage diseases ͉ adeno-associated virus N iemann-Pick type A disease (NPA) is a lysosomal storage disorder caused by a deficiency in acid sphingomyelinase (ASM) activity. Loss of ASM activity results in lysosomal sphingomyelin (SPM) accumulation, secondary metabolic defects such as aberrant cholesterol metabolism, and subsequently the loss of cellular function in various organ systems including the central nervous system (CNS) (1, 2). Previously, we demonstrated that intracerebral delivery of adeno-associated virus serotype-2 encoding human ASM (AAV2-hASM) is effective in correcting lysosomal storage pathology in ASM knockout (ASMKO) mice (3). Interestingly, correction of lysosomal storage pathology not only occurred at the injection site (i.e., hippocampus), but also in regions (e.g., entorhinal cortex) that send and͞or receive input from the injection site. This finding, in accordance with previous work (4), demonstrated that neuronal circuits can be used to achieve AAV vector and protein distribution via axonal transport to regions that are proximal or distal to the injection site.Despite our initial success with AAV2, investigation of the relative therapeutic efficacy of additional serotypes is warranted. Serotype-dependent variations in cellular tropism, rates of diffusion, and transduction efficiencies may translate into disparate levels of therapeutic efficacy (5, 6). Furthermore, we wish to show that AAV-mediated hASM expression prevents disease initiated neurodegeneration (i.e., Purkinje cell loss) and disturbances in motor function in ASMKO mice (7, §).To examine...

The possible contribution of HLA-DRB3, -DRB4, and -DRB5 alleles to type 1 diabetes risk and to insulin autoantibody (IAA), GAD65 (GAD autoantibody [GADA]), IA-2 antigen (IA-2A), or ZnT8 against either of the three amino acid variants R, W, or Q at position 325 (ZnT8RA, ZnT8WA, and ZnT8QA, respectively) at clinical diagnosis is unclear. Next-generation sequencing (NGS) was used to determine all DRB alleles in consecutively diagnosed patients ages 1–18 years with islet autoantibody–positive type 1 diabetes (n = 970) and control subjects (n = 448). DRB3, DRB4, or DRB5 alleles were tested for an association with the risk of DRB1 for autoantibodies, type 1 diabetes, or both. The association between type 1 diabetes and DRB1*03:01:01 was affected by DRB3*01:01:02 and DRB3*02:02:01. These DRB3 alleles were associated positively with GADA but negatively with ZnT8WA, IA-2A, and IAA. The negative association between type 1 diabetes and DRB1*13:01:01 was affected by DRB3*01:01:02 to increase the risk and by DRB3*02:02:01 to maintain a negative association. DRB4*01:03:01 was strongly associated with type 1 diabetes (P = 10−36), yet its association was extensively affected by DRB1 alleles from protective (DRB1*04:03:01) to high (DRB1*04:01:01) risk, but its association with DRB1*04:05:01 decreased the risk. HLA-DRB3, -DRB4, and -DRB5 affect type 1 diabetes risk and islet autoantibodies. HLA typing with NGS should prove useful to select participants for prevention or intervention trials.