Disposition of the herbicide 2-chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine (Atrazine) and its major metabolites in mice: a liquid chromatography/mass spectrometry analysis of urine, plasma, and tissue levels.

2-Chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine (atrazine, ATR) is a toxicologically important and widely used herbicide. Recent studies have shown that it can elicit neurological, immunological, developmental, and biochemical alterations in several model organisms, including in mice. Because disposition data in mice are lacking, we evaluated ATR's metabolism and tissue dosimetry after single oral exposures (5-250 mg/kg) in C57BL/6 mice using liquid chromatography/mass spectrometry (Ross and Filipov, 2006). ATR was metabolized and cleared rapidly; didealkyl ATR (DACT) was the major metabolite detected in urine, plasma, and tissues. Plasma ATR peaked at 1 h postdosing and rapidly declined, whereas DACT peaked at 2 h and slowly declined. Most ATR and metabolite residues were excreted within the first 24 h. However, substantial amounts of DACT were still present in 25- to 48-h and 49- to 72-h urine. ATR reached maximal brain levels (0.06-1.5 microM) at 4 h (5-125 mg/kg) and 1 h (250 mg/kg) after dosing, but levels quickly declined to <0.1 microM by 12 h in all the groups. In contrast, strikingly high concentrations of DACT (1.5-50 microM), which are comparable with liver DACT levels, were detectable in brain at 2 h. Brain DACT levels slowly declined, paralleling the kinetics of plasma DACT. Our findings suggest that in mice ATR is widely distributed and extensively metabolized and that DACT is a major metabolite detected in the brain at high levels and is ultimately excreted in urine. Our study provides a starting point for the establishment of models that link target tissue dose to biological effects caused by ATR and its in vivo metabolites.

The sensitization of a broad spectrum of sensory nerve fibers in a rat model of acute postoperative pain and its response to intrathecal pharmacotherapy.

Further understanding of pathophysiology of postoperative acute pain is necessary for its better management. The methodology of current threshold (CT) determination by using sine-wave stimuli at 3 frequencies has been used to selectively and quantitatively analyze the function of the subsets of fibers (i.e., frequency of 5, 250, and 2000Hz recruits C-, Adelta-, and Abeta-fibers, respectively). This study investigated how surgical incision would affect the CTs, and then assessed the efficacy of intrathecal pharmacotherapy. The CT required to evoke a paw withdrawal response was assessed over time at stimulus frequencies of 5Hz (CT5), 250Hz (CT250), and 2000Hz (CT2000) in rats that had undergone surgical incision of the plantar skin and muscle. The CTs at all frequencies significantly decreased immediately after the incision. The decreased thresholds gradually recovered during the first week post-surgery. CT5 and CT250 (but not CT2000) remained significantly low even on day 7 post-surgery. Morphine at 5microg/10microL i.t. significantly reversed CT5 and CT250. NBQX (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid [AMPA]/kainate receptor antagonist) at 1.9 or 3.8microg/10microL i.t. significantly increased the thresholds over the pre-surgery threshold levels at all frequencies. MK-801 (N-methyl d-aspartate [NMDA] receptor antagonist) up to 13.5microg/10microL i.t. did not significantly affect CTs at any frequencies. In conclusion, a broad spectrum of sensory fibers (Abeta, Adelta, and C) is sensitized at the spinal and/or peripheral level in the postoperative acute pain state. Spinal AMPA/kainate receptors but not NMDA receptors play a significant role in this sensitization.

Secondary hyperalgesia in the postoperative pain model is dependent on spinal calcium/calmodulin-dependent protein kinase II alpha activation.

BACKGROUND: Spinally administered non-N-methyl-D-aspartate (NMDA), but not NMDA, receptor antagonists block primary (1 degree) and secondary (2 degrees) mechanical hyperalgesia and spontaneous pain after plantar incision. Hyperalgesia after thermal stimulation is also mediated by non-NMDA, but not NMDA, receptors. Although previous pain behavior studies in the thermal stimulus model demonstrated distinct protein kinase involvement downstream from spinal non-NMDA receptor activation, protein kinase signaling mechanisms have not been examined in the postoperative pain model. In the present study, we investigated whether spinal calcium/calmodulin-dependent protein kinase IIalpha (CaMKIIalpha) mediates 1 degree and/or 2 degrees hyperalgesia and spontaneous pain behavior after plantar incision. METHODS: Catheterized rats received a 1 cm incision in the hindpaw and were tested over 2 days for responses to mechanical stimulation adjacent to or 1 cm away from the incision site. Some rats received intrathecal (IT) pretreatment with a CaMKIIalpha inhibitor (14, 34, or 104 nmol KN-93) or vehicle (5% dimethyl sulfoxide in sterile saline). Separate groups received IT 34 nmol or 104 nmol KN-93 and were tested for hindpaw weight bearing. Lumbar spinal cords were extracted 1 h after incision or sham treatment to measure phosphorylated CaMKIIalpha and alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid GLUR1-831 in Western immunoblots. RESULTS: Incision increased spinal CaMKIIalpha and GLUR1-831 phosphorylation. Although pretreatment with all doses of IT KN-93 reduced the development of 2 degrees hyperalgesia, only 34 nmol KN-93 appeared to have an effect on 1 degrees hyperalgesia. IT KN-93 did not affect nonevoked pain. CONCLUSION: Spinal sensitization underlying incision-evoked hyperalgesia involves spinal CaMKIIalpha activation and enhanced spinal alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid receptor (AMPA) function.

Low-speed subcellular fractionation method for determining noxious stimulus-evoked spinal neurokinin-1 receptor internalization.

Substance P release from nociceptive primary afferents activates post-synaptic neurokinin-1 (NK-1) receptors causing subsequent NK-1 receptor internalization. Fluorescent immunohistochemistry is typically used to quantify NK-1 receptor internalization, an indirect measure of substance P (SP) release. However, this technique entails several limitations that restrict its application. Using simple subcellular fractionation and immunoblotting methods, we demonstrate that intrathecal SP invokes a rapid and dose-dependent increase in dorsal horn cytoplasmic NK-1 receptors. We also show that hind paw compression and noxious thermal stimulation increase cytoplasmic NK-1 receptor, when compared to sham stimulations. Fluorescent immunohistochemistry confirmed that increases in cytoplasmic NK-1 corresponded with increased NK-1 receptor internalization. Herein, we report that low-speed centrifugation and Western immunoblotting provide NK-1 internalization results consistent with those obtained by more traditional methods. These data support previous findings demonstrating a role for spinal NK-1 receptors in nociceptive processing.

Regulation of peripheral inflammation by spinal p38 MAP kinase in rats.

BACKGROUND: Somatic afferent input to the spinal cord from a peripheral inflammatory site can modulate the peripheral response. However, the intracellular signaling mechanisms in the spinal cord that regulate this linkage have not been defined. Previous studies suggest spinal cord p38 mitogen-activated protein (MAP) kinase and cytokines participate in nociceptive behavior. We therefore determined whether these pathways also regulate peripheral inflammation in rat adjuvant arthritis, which is a model of rheumatoid arthritis. METHODS AND FINDINGS: Selective blockade of spinal cord p38 MAP kinase by administering the p38 inhibitor SB203580 via intrathecal (IT) catheters in rats with adjuvant arthritis markedly suppressed paw swelling, inhibited synovial inflammation, and decreased radiographic evidence of joint destruction. The same dose of SB203580 delivered systemically had no effect, indicating that the effect was mediated by local concentrations in the neural compartment. Evaluation of articular gene expression by quantitative real-time PCR showed that spinal p38 inhibition markedly decreased synovial interleukin-1 and -6 and matrix metalloproteinase (MMP3) gene expression. Activation of p38 required tumor necrosis factor alpha (TNFalpha) in the nervous system because IT etanercept (a TNF inhibitor) given during adjuvant arthritis blocked spinal p38 phosphorylation and reduced clinical signs of adjuvant arthritis. CONCLUSIONS: These data suggest that peripheral inflammation is sensed by the central nervous system (CNS), which subsequently activates stress-induced kinases in the spinal cord via a TNFalpha-dependent mechanism. Intracellular p38 MAP kinase signaling processes this information and profoundly modulates somatic inflammatory responses. Characterization of this mechanism could have clinical and basic research implications by supporting development of new treatments for arthritis and clarifying how the CNS regulates peripheral immune responses.

Covariance among age, spinal p38 MAP kinase activation and allodynia.

UNLABELLED: This study examined effects of age (young rats, approximately 35 days, vs mature rats, approximately 75-110 days) on spinal nerve ligation (SNL)-induced tactile allodynia and phosphorylation of p38 (as measured by phospho-p38 MAP kinase [P-p38]) in dorsal root ganglia and spinal cord. Effects of SNL combined with spinal nerve transection also were assessed. Mature rats displayed milder SNL-induced allodynia than young rats. Addition of spinal nerve transection distal to the ligation in older animals resulted in an allodynia comparable to that seen in young animals. In DRG, both groups displayed early (5 h) and late (10 days) peaks in P-p38 following surgery as compared to naïve rats. Tight nerve ligation plus transection had no additional effect on P-p38 levels in DRG. In spinal cord, young rats had increased levels of P-p38 from 5 h to 3 days after SNL. Phosphorylated p38 levels then decreased, with a second peak at 10 days. In contrast, spinal cord from mature rats showed less early p38 phosphorylation, although they also displayed a late 10-day peak. Addition of a transection to the ligation produced restoration of the early peak along with intensification of allodynia. Alterations of spinal P-p38 at early time points thus seem to covary with intensity of tactile allodynia. PERSPECTIVE: Age and modifications to spinal nerve ligation, a common model of neuropathic pain, influence spinal p38 phosphorylation and allodynia. Early levels of spinal P-p38 seem to covary with allodynia intensity. This may mean that small variations of an injury could affect the therapeutic window of a p38 antagonist.

Activated PKA and PKC, but not CaMKIIalpha, are required for AMPA/Kainate-mediated pain behavior in the thermal stimulus model.

Secondary mechanical allodynia resulting from a thermal stimulus (52.5 degrees C for 45s) is blocked by intrathecal (i.t.) pretreatment with calcium-permeable AMPA/KA receptor antagonists, but not NMDA receptor antagonists. Spinal sensitization is presumed to underlie thermal stimulus-evoked secondary mechanical allodynia. We investigated whether this spinal sensitization involves activation and phosphorylation of calcium-dependent protein kinases (PKA, PKC and CaMKIIalpha), and examined if the noxious stimulus increases phosphorylated AMPA GLUR1 (pGLUR1 Ser-845 and pGLUR1 Ser-831). Secondary mechanical allodynia after thermal stimulation was not altered by i.t. pretreatment with control vehicles (saline or 5% DMSO). Comparable allodynia was observed after pretreatment with a selective CaMKIIalpha inhibitor (17 and 34nmol KN-93). In marked contrast, pretreatment with either a PKA (10nmol H89) or PKC (30nmol chelerythrine) inhibitor blocked allodynia. Western immunoblot analyses supported behavioral findings and revealed a thermal stimulus-evoked increase in spinal phosphorylated PKA and PKC, but not CaMKIIalpha. There was no increase in any of the total protein kinases. Although thermal stimulation did not change either pGLUR1 Ser-845 or pGLUR1 Ser-831, it was associated with an increase in cytosolic total GLUR1. Pretreatment with a selective calcium-permeable AMPA/KA receptor antagonist (5nmol joro spider toxin), but not an NMDA receptor antagonist (25nmol d-2-amino-5-phosphonovalerate, AP-5), blocked thermal stimulus-evoked increases in phosphorylated PKA and PKC, in addition to increased cytosolic GLUR1. These findings indicate that spinal sensitization in the thermal stimulus model does not involve CaMKIIalpha activation or AMPA GLUR1 receptor phosphorylation, and differs from that occurring in NMDAr-dependent pain states.

GABAB receptors on central terminals of C-afferents mediate intersegmental Adelta-afferent evoked hypoalgesia.

The current study tested the hypothesis that repetitive activation of sciatic Adelta-afferents evokes a saphenous C-afferent hypoalgesia mediated by pre-synaptic GABA(B) receptors. Tonic activation of sciatic Adelta-afferents was produced by cutaneous application of dimethyl sulfoxide (DMSO) followed by repetitive thermal activation of Adelta-afferents on the dorsolateral hind paw. The tonic activation of sciatic Adelta-afferents produced hypoalgesia in saphenous C-afferents. Intrathecal administration of the GABA(B) receptor antagonist, saclofen, attenuated saphenous hypoalgesia demonstrating at least partial mediation by central GABA(B) receptors. To determine if this central GABA(B) receptor activation occurs at pre-synaptic primary afferent terminals or postsynaptic spinal cord neurons, the dorsal hind paws of mice were infected with a recombinant herpes simplex virus type 1 (HSV-1) designed to selectively knock down expression of the GABA(B1a) receptor subunit (PAGB1a) in primary afferents or a control virus encoding the E. coli lacZ gene (PZ). Four weeks after infection, GABA(B) receptor immunoreactivity in the superficial dorsal horns ipsilateral to PAGB1a application was reduced and hypoalgesia in saphenous C-afferents was attenuated when compared to PZ-infected mice. These findings indicate an intersegmental, sciatic Adelta-afferent-evoked hypoalgesic effect on saphenous C-afferent responses that is mediated by pre-synaptic GABA(B) receptors on the terminals of those C-afferents.

Spinal blockade of TNF blocks spinal nerve ligation-induced increases in spinal P-p38.

Spinal nerve ligation (SNL) results in a profound long lasting allodynia and increases in phosphorylated p38 in dorsal root ganglia (DRG) neurons and spinal cord microglia. We have previously shown that systemic etanercept, a tumor necrosis factor (TNF) antagonist, reduced allodynia by 42% and blocked SNL-induced increases in P-p38 levels in the L5 and L6 DRG, but not in the ipsilateral lumbar spinal cord. The present experiments demonstrated that intrathecal etanercept (100 microg) prevents SNL-induced increased levels of spinal P-p38. Pretreatment, but not posttreatment, with intrathecal etanercept (100 microg), given every third day, reduced mechanical allodynia by 50%. This therapeutic benefit was maintained for at least 7 days after cessation of treatment. Combined systemic and intrathecal administration of etanercept was no more effective than intrathecal treatment alone. These data imply that TNF provides the trigger for phosphorylation of p38 in both DRG neurons and spinal microglia.

Calcium-permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate receptors mediate development, but not maintenance, of secondary allodynia evoked by first-degree burn in the rat.

Intrathecal pretreatment with N-methyl-D-aspartate (NMDA) receptor antagonists blocks development of spinal sensitization in a number of pain models. In contrast, secondary mechanical allodynia evoked by thermal injury (52.5 degrees C for 45 s) applied to the hind paw of the rat is not blocked by intrathecal pretreatment with NMDA receptor antagonists. It is, however, blocked by antagonists to the non-NMDA, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate (AMPA/KA) and calcium-permeable AMPA/KA receptors. These findings suggest a role for these receptors in the development of spinal sensitization. The present study used the same thermal injury model to assess the effects of the AMPA/KA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and specific calcium-permeable AMPA/KA receptor antagonists philanthotoxin (PHTx) and joro spider toxin (JST) when given as postinjury treatments. Intrathecal saline injection at 5 and 30 min postinjury had no effect on thermal injury-evoked allodynia as measured by calibrated von Frey filaments. In contrast, 36 nmol of CNQX given at either time point reversed allodynia. Intrathecal 13 nmol of PHTx or 9 nmol of JST (higher doses than that required for pretreatment) reversed allodynia at the 5-min time point, but neither drug was antiallodynic at the 30-min time point. Thus, secondary mechanical allodynia in this model is not maintained by calcium-permeable AMPA/KA receptors, but instead requires activation of calcium-impermeable AMPA/KA receptors. This finding supports a role for AMPA/KA receptor function in responses occurring during spinal sensitization.

Afferent fiber-selective shift in opiate potency following targeted opioid receptor knockdown.

Spinal application of opiates is the cornerstone of potent analgesia. In the present study, opiate analgesia was investigated after cutaneous application of a recombinant herpes simplex virus type-1 (HSV-1) encoding micro-opioid receptor (microOR) cDNA in reverse orientation with respect to the human cytomegalovirus early enhancer-promoter. Hind paw application of this recombinant vector was used in order to attenuate expression of the microOR in primary afferents and determine whether recombinant vector application would differentially affect the antinociceptive effects of the specific microOR agonist, [D-Ala(2),N-MePhe(4),Gly-ol(5)] enkephalin (DAMGO), on behavioral responses mediated by C- and Adelta-thermonociceptors. The recombinant vector encoding the Escherichia coli lacZ gene marker, KHZ, served as a control virus. Dorsal hind paw surfaces of female Swiss-Webster mice were treated with one of these two viruses (1x10(8)pfu, 10 microl) or vehicle (uninfected). Immunohistochemistry and quantitative image analyses revealed decreased microOR expression in the superficial dorsal horns ipsilateral to hind paws treated with AMOR, but not KHZ. To add, behavioral foot withdrawal latencies of AMOR- and KHZ-treated hind paws demonstrated dose-dependent antinociception after intrathecal DAMGO administration. However, cutaneous application of dorsal hind paw surfaces treated with AMOR, but not KHZ, caused a rightward shift in the C-fiber dose-response, thus, indicating a loss of potency of intrathecal DAMGO. Loss or diminution of DAMGO potency during Adelta-fiber-mediated responses was not observed. These immunohistochemistry and behavioral results of novel, recombinant HSV-1 vector microOR 'knock-down' in nociceptor afferent fibers provide additional evidence for presynaptic localization of microORs on central C-, but not Adelta-terminals.