Inflammation-induced GluA1 trafficking and membrane insertion of Ca2+ permeable AMPA receptors in dorsal horn neurons is dependent on spinal tumor necrosis factor, PI3 kinase and protein kinase A.

Peripheral inflammation induces sensitization of nociceptive spinal cord neurons. Both spinal tumor necrosis factor (TNF) and neuronal membrane insertion of Ca2+ permeable AMPA receptor (AMPAr) contribute to spinal sensitization and resultant pain behavior, molecular mechanisms connecting these two events have not been studied in detail. Intrathecal (i.t.) injection of TNF-blockers attenuated paw carrageenan-induced mechanical and thermal hypersensitivity. Levels of GluA1 and GluA4 from dorsal spinal membrane fractions increased in carrageenan-injected rats compared to controls. In the same tissue, GluA2 levels were not altered. Inflammation-induced increases in membrane GluA1 were prevented by i.t. pre-treatment with antagonists to TNF, PI3K, PKA and NMDA. Interestingly, administration of TNF or PI3K inhibitors followed by carrageenan caused a marked reduction in plasma membrane GluA2 levels, despite the fact that membrane GluA2 levels were stable following inhibitor administration in the absence of carrageenan. TNF pre-incubation induced increased numbers of Co2+ labeled dorsal horn neurons, indicating more neurons with Ca2+ permeable AMPAr. In parallel to Western blot results, this increase was blocked by antagonism of PI3K and PKA. In addition, spinal slices from GluA1 transgenic mice, which had a single alanine replacement at GluA1 ser 845 or ser 831 that prevented phosphorylation, were resistant to TNF-induced increases in Co2+ labeling. However, behavioral responses following intraplantar carrageenan and formalin in the mutant mice were no different from littermate controls, suggesting a more complex regulation of nociception. Co-localization of GluA1, GluA2 and GluA4 with synaptophysin on identified spinoparabrachial neurons and their relative ratios were used to assess inflammation-induced trafficking of AMPAr to synapses. Inflammation induced an increase in synaptic GluA1, but not GluA2. Although total GluA4 also increased with inflammation, co-localization of GluA4 with synaptophysin, fell short of significance. Taken together these data suggest that peripheral inflammation induces a PI3K and PKA dependent TNFR1 activated pathway that culminates with trafficking of calcium permeable AMPAr into synapses of nociceptive dorsal horn projection neurons.

Mitotoxicity and bortezomib-induced chronic painful peripheral neuropathy.

Many of the most effective anti-cancer drugs induce a dose-limiting peripheral neuropathy that compromises therapy. Evidence from animal models of chemotherapy-induced painful peripheral neuropathy produced by the taxane agent, paclitaxel, and the platinum-complex agent, oxaliplatin, indicate that they produce neuropathy via a common mechanism-a toxic effect on the mitochondria in primary afferent sensory neurons. Bortezomib is from the proteasome-inhibitor class of chemotherapeutics. It also produces a dose-limiting peripheral neuropathy, but its effects on neuronal mitochondria are unknown. To investigate this, we developed a model of bortezomib-induced painful peripheral neuropathy in the rat and assessed mitochondrial function (respiration and ATP production) in sciatic nerve samples harvested at two time points: day 7, which is three days after treatment and before pain appears, and day 35, which is one month post-treatment and the time of peak pain severity. We found significant deficits in Complex I-mediated and Complex II-mediated respiration, and in ATP production at both time points. Prophylactic treatment with acetyl-L-carnitine, which has previously been shown to prevent paclitaxel- and oxaliplatin-induced mitochondrial dysfunction and pain, completely blocked bortezomib's effects on mitochondria and pain. These results suggest that mitotoxicity may be the core pathology for all chemotherapy-induced peripheral neuropathy and that drugs that protect mitochondrial function may be useful chemotherapy adjuncts.

Characterization of oxaliplatin-induced chronic painful peripheral neuropathy in the rat and comparison with the neuropathy induced by paclitaxel.

Anti-neoplastic agents in the platinum-complex, taxane, vinca alkaloid, and proteasome-inhibitor classes induce a dose-limiting, chronic, distal, symmetrical, sensory peripheral neuropathy that is often accompanied by neuropathic pain. Clinical descriptions suggest that these conditions are very similar, but clinical data are insufficient to determine the degree of similarity and to determine if they share common pathophysiological mechanisms. Animal models do not have the limitations of clinical studies and so we have characterized a rat model of chronic painful peripheral neuropathy induced by a platinum-complex agent, oxaliplatin, in order to compare it with a previously characterized model of chronic painful peripheral neuropathy induced by a taxane agent, paclitaxel. The oxaliplatin model evokes mechano-allodynia, mechano-hyperalgesia, and cold-allodynia that have a delayed onset, gradually increasing severity, a distinct delay to peak severity, and duration of about 2.5 months. There is no effect on heat sensitivity. Electron microscopy (EM) analyses found no evidence for axonal degeneration in peripheral nerve, and there is no upregulation of activating transcription factor-3 in the lumbar dorsal root ganglia. There is a statistically significant loss of intraepidermal nerve fibers in the plantar hind paw skin. Oxaliplatin treatment causes a significant increase in the incidence of swollen and vacuolated mitochondria in peripheral nerve axons, but not in their Schwann cells. Nerve conduction studies found significant slowing of sensory axons, but no change in motor axons. Single fiber recordings found an abnormal incidence of A- and C-fibers with irregular, low-frequency spontaneous discharge. Prophylactic dosing with two drugs that are known to protect mitochondria, acetyl-l-carnitine and olesoxime, significantly reduced the development of pain hypersensitivity. Our results are very similar to those obtained previously with paclitaxel, and support the hypothesis that these two agents, and perhaps other chemotherapeutics, produce very similar conditions because they have a mitotoxic effect on primary afferent neurons.

Mitochondrial abnormality in sensory, but not motor, axons in paclitaxel-evoked painful peripheral neuropathy in the rat.

The dose-limiting side effect of the anti-neoplastic agent, paclitaxel, is a chronic distal symmetrical peripheral neuropathy that produces sensory dysfunction (hypoesthesia and neuropathic pain) but little or no distal motor dysfunction. Similar peripheral neuropathies are seen with chemotherapeutics in the vinca alkaloid, platinum-complex, and proteasome inhibitor classes. Studies in rats suggest that the cause is a mitotoxic effect on axonal mitochondria. If so, then the absence of motor dysfunction may be due to mitotoxicity that affects sensory axons but spares motor axons. To investigate this, paclitaxel exposure levels in the dorsal root, ventral root, dorsal root ganglion, peripheral nerve, and spinal cord were measured, and the ultrastructure and the respiratory function of mitochondria in dorsal roots and ventral roots were compared. Sensory and motor axons in the roots and nerve had comparably low exposure to paclitaxel and exposure in the spinal cord was negligible. However, sensory neurons in the dorsal root ganglion had a very high and remarkably persistent (up to 10 days or more after the last injection) exposure to paclitaxel. Paclitaxel evoked a significant increase in the incidence of swollen and vacuolated mitochondria in the myelinated and unmyelinated sensory axons of the dorsal root (as seen previously in the peripheral nerve) but not in the motor axons of the ventral root. Stimulated mitochondrial respiration in the dorsal root was significantly depressed in paclitaxel-treated animals examined 2-4 weeks after the last injection, whereas respiration in the ventral root was normal. We conclude that the absence of motor dysfunction in paclitaxel-evoked peripheral neuropathy may be due to the absence of a mitotoxic effect in motor neuron axons, whereas the sensory dysfunction may be due to a mitotoxic effect resulting from the primary afferent neuron's cell body being exposed to high and persistent levels of paclitaxel.

Randomized clinical trial to evaluate the efficacy of a 5-day ceftiofur hydrochloride intramammary treatment on nonsevere gram-negative clinical mastitis.

The objective of this study was to evaluate the efficacy of intramammary treatment with ceftiofur hydrochloride of nonsevere, clinical coliform mastitis. One hundred four cases on 5 farms met the enrollment criteria for the study. Escherichia coli was the most common coliform species identified in milk samples from cows with mild to moderate clinical mastitis, followed by Klebsiella spp. and Enterobacter spp. At enrollment, a milk sample from the affected quarter was taken and used for on-farm culture or submitted to the laboratory. For cows in the treatment group, treatment was initiated with ceftiofur hydrochloride via intramammary infusion at 24-h intervals for 5 d according to label standards. Cows in the control group did not receive treatment. Culture results were available on the day after enrollment and only cows with coliform mastitis continued in the treatment and untreated control groups. Bacteriological cure was defined based on 2 posttreatment milk samples. Molecular typing was used for final definition of bacteriological cure. Treatment of nonsevere clinical gram-negative mastitis with ceftiofur hydrochloride resulted in a significant increase in bacteriological cure compared with nontreated controls in animals infected with E. coli or Klebsiella spp. Treated animals clinically improved significantly more compared with control cows. No significant differences were observed between treated and control animals in milk production or linear score before or after clinical mastitis. Treated animals left the study less frequently compared with control animals.

The effect of recurrent episodes of clinical mastitis caused by gram-positive and gram-negative bacteria and other organisms on mortality and culling in Holstein dairy cows.

The objective of this study was to estimate the effects of recurrent episodes of different types of clinical mastitis (CM) caused by gram-positive (Streptococcus spp., Staphylococcus aureus, Staphylococcus spp.) and gram-negative (Escherichia coli, Klebsiella, Citrobacter, Enterobacter, Pseudomonas) bacteria, and other organisms (Arcanobacterium pyogenes, Mycoplasma, Corynebacterium bovis, yeast, miscellaneous) on the probability of mortality and culling in Holstein dairy cows. Data from 30,233 lactations in cows of 7 dairy farms in New York State were analyzed. Cows were followed for the first 10 mo in lactation, or until death or culling occurred, or until the end of our study period. Generalized linear mixed models with a Poisson error distribution were used to study the effects of recurrent cases of the different types of CM and several other factors (herd, parity, month of lactation, current year and season, profitability, net replacement cost, other diseases) on cows' probability of death (model 1) or being culled (model 2). Primiparous and multiparous cows were modeled separately because they had different risks of mortality and culling and potentially different CM effects on mortality and culling. Approximately 30% of multiparous cows had at least one case of CM in lactation compared with 16.6% of primiparous cows. Multipara also had higher lactational incidence risks of second (10.7%) and third (4.4%) cases than primipara (3.7% and 1.1%, respectively). For primipara, CM increased the probability of death, with each successive case occurring in a month being increasingly lethal. In multipara, gram-negative CM increased the probability of death, especially when the gram-negative case was the first or second CM case in lactation. Primiparous cows with CM were more likely to be culled after CM than if they did not have CM, particularly after a second or third case. In multipara, any type of CM increased the probability of being culled. Gram-negative CM cases were associated with the numerically highest risk of culling.

Sources of Klebsiella and Raoultella species on dairy farms: be careful where you walk.

Klebsiella spp. are a common cause of mastitis, milk loss, and culling on dairy farms. Control of Klebsiella mastitis is largely based on prevention of exposure of the udder to the pathogen. To identify critical control points for mastitis prevention, potential Klebsiella sources and transmission cycles in the farm environment were investigated, including oro-fecal transmission, transmission via the indoor environment, and transmission via the outdoor environment. A total of 305 samples was collected from 3 dairy farms in upstate New York in the summer of 2007, and included soil, feed crops, feed, water, rumen content, feces, bedding, and manure from alleyways and holding pens. Klebsiella spp. were detected in 100% of rumen samples, 89% of water samples, and approximately 64% of soil, feces, bedding, alleyway, and holding pen samples. Detection of Klebsiella spp. in feed crops and feed was less common. Genotypic identification of species using rpoB sequence data showed that Klebsiella pneumoniae was the most common species in rumen content, feces, and alleyways, whereas Klebsiella oxytoca, Klebsiella variicola, and Raoultella planticola were the most frequent species among isolates from soil and feed crops. Random amplified polymorphic DNA-based strain typing showed heterogeneity of Klebsiella spp. in rumen content and feces, with a median of 4 strains per 5 isolates. Observational and bacteriological data support the existence of an oro-fecal transmission cycle, which is primarily maintained through direct contact with fecal contamination or through ingestion of contaminated drinking water. Fecal shedding of Klebsiella spp. contributes to pathogen loads in the environment, including bedding, alleyways, and holding pens. Hygiene of alleyways and holding pens is an important component of Klebsiella control on dairy farms.

The response of spinal microglia to chemotherapy-evoked painful peripheral neuropathies is distinct from that evoked by traumatic nerve injuries.

Painful peripheral neuropathies produced by nerve trauma are accompanied by substantial axonal degeneration and by a response in spinal cord microglia that is characterized by hypertrophy and increased expression of several intracellular and cell-surface markers, including ionizing calcium-binding adapter molecule 1 (Iba1) and Cd11b (a complement receptor 3 antigen recognized by the OX42 antibody). The microglia response has been hypothesized to be essential for the pathogenesis of the neuropathic pain state. In contrast, the painful peripheral neuropathies produced by low doses of cancer chemotherapeutics do not produce degeneration of axons in the peripheral nerve, although they do cause partial degeneration of the sensory axons' distal-most tips, that is the intraepidermal nerve fibers that form the axons' terminal receptor arbors. The question thus arises as to whether the relatively minor and distal axonal injury characterizing the chemotherapy-evoked neuropathies is sufficient to evoke the microglial response that is seen after traumatic nerve injury. We examined the lumbar spinal cord of rats with painful peripheral neuropathies due to the anti-neoplastic agents, paclitaxel, vincristine, and oxaliplatin, and the anti-retroviral agent, 2',3'-dideoxycytidine (ddC), and compared them to rats with a complete sciatic nerve transection and the partial sciatic nerve injury produced in the chronic constriction injury model (CCI). As expected, microglia hypertrophy and increased expression of Iba1 were pronounced in the nerve transection and CCI animals. However, there was no microglia hypertrophy or increased Iba1 staining in the animals treated with paclitaxel, vincristine, oxaliplatin, or ddC. These results suggest that the mechanisms that produce neuropathic pain after exposure to chemotherapeutics may be fundamentally different than those operating after nerve trauma.

Effects of clinical mastitis caused by gram-positive and gram-negative bacteria and other organisms on the probability of conception in New York State Holstein dairy cows.

The objective of this study was to estimate the effects of different types of clinical mastitis (CM) on the probability of conception in New York State Holstein cows. Data were available on 55,372 artificial inseminations (AI) in 23,695 lactations from 14,148 cows in 7 herds. We used generalized linear mixed models to model whether or not a cow conceived after a particular AI. Independent variables included AI number (first, second, third, fourth), parity, season when AI occurred, farm, type of CM (due to gram-positive bacteria, gram-negative bacteria, or other organisms) in the 6 wk before and after an AI, and occurrence of other diseases. Older cows were less likely to conceive. Inseminations occurring in the summer were least likely to be successful. Retained placenta decreased the probability of conception. Conception was also less likely with each successive AI. The probability of conception associated with the first AI was 0.29. The probability of conception decreased to 0.26, 0.25, and 0.24 for the second, third, and fourth AI, respectively. Clinical mastitis occurring any time between 14 d before until 35 d after an AI was associated with a lower probability of conception; the greatest effect was an 80% reduction associated with gram-negative CM occurring in the week after AI. In general, CM due to gram-negative bacteria had a more detrimental effect on probability of conception than did CM caused by gram-positive bacteria or other organisms. Furthermore, CM had more effect on probability of conception immediately around the time of AI. Additional information about CM (i.e., its timing with respect to AI, and whether the causative agent is gram-positive or gram-negative bacteria, or other organisms) is valuable to dairy personnel in determining why some cows are unable to conceive in a timely manner. These findings are also beneficial for the management of mastitic cows (especially those with gram-negative CM) when mastitis occurs close to AI.

Effects of repeated gram-positive and gram-negative clinical mastitis episodes on milk yield loss in Holstein dairy cows.

The objective of this study was to estimate the effects of recurrent episodes of gram-positive and gram-negative cases of clinical mastitis (CM) on milk production in Holstein dairy cows. We were interested in the severity of repeated cases in general, but also in the severity of the host response as judged by milk production loss when a previous case was caused by a similar or different microorganism. The results were based on data from 7,721 primiparous lactations and 13,566 multiparous lactations in 7 large dairy herds in New York State. The distribution of organisms in the CM cases showed 28.5% gram-positive cases, 31.8% gram-negative cases, 15.0% others, and 24.8% with no organism identified. Mixed models, with a random herd effect and an autoregressive covariance structure to account for repeated measurements, were used to quantify the effect of repeated CM and several other control variables (parity, week of lactation, other diseases) on milk yield. Our data indicated that repeated CM cases showed a very similar milk loss compared with the first case. No reduction of severity was present with increasing count of the CM case. Gram-negative cases had more severe milk loss compared with gram-positive and other cases irrespective of the count of the case in lactation. Milk loss in multipara (primipara) due to gram-negative CM was approximately 304 kg (228 kg) in the 50 d following CM. This loss was approximately 128 kg (133 kg) for gram-positive cases and 92 kg (112 kg) for other cases. The severity of a second case of gram-negative CM was not reduced by previous cases of gram-negative CM in multipara and only slightly less severe in a similar scenario in primipara cows. Similarly, a previous gram-positive case did not reduce severity of a second or third gram-positive case. Hence, our data do not support that immunological memory of previous exposure to an organism in the same generic class provides protection for a next case of CM with an organism in the same class.

De novo expression of the neurokinin 1 receptor in spinal lamina I pyramidal neurons in polyarthritis.

Spinal lamina I (LI) neurons play a major role in the transmission and integration of pain-related information that is relayed to higher centers. Alterations in the excitability of these neurons influence chronic pain development, and expression of the neurokinin 1 receptor (NK-1r) is thought to play a major role in such changes. Novel expression of NK-1r may underlie hyperexcitability in new populations of LI neurons. LI projection neurons can be classified morphologically into fusiform, pyramidal, and multipolar cells, differing in their functional properties, with the pyramidal type being nonnociceptive. In agreement with this, we have shown that spinoparabrachial pyramidal neurons seldom express NK-1r, in contrast with the other two cell types. In this study we investigated in the rat the long-term changes in NK-1r expression by spinoparabrachial LI neurons following the unilateral injection in the hindpaw plantar surface of complete Freund's adjuvant (CFA). Cholera toxin subunit B (CTb) was injected unilaterally into the parabrachial nucleus. Our results revealed that, ipsilaterally, pyramidal neurons were seldom immunoreactive for NK-1r both in saline-injected and in CFA-injected rats, up to 10 days post-CFA. However, a considerable number of pyramidal cells were immunoreactive for NK-1r at 15, 21, and 30 days post-CFA. Our data raise the possibility -- which needs to be confirmed by electrophysiology -- that most LI projection neurons of the pyramidal type are likely nonnociceptive in naive animals but might become nociceptive following the development of arthritis.

Milk production change following clinical mastitis and reproductive performance compared among J5 vaccinated and control dairy cattle.

Naturally occurring cases of bovine clinical mastitis (CM) were studied among J5 vaccinates and controls on 3 commercial dairy farms. Milk production change and reproductive performance following CM were compared between the 2 groups. Among 306 controls and 251 vaccinates, there were 221 new cases of CM affecting 120 cows; 437 lactations never had a case of CM. Environmental pathogens made up 90% (159/176) of etiologic agents isolated. Change in daily milk production following CM was associated with J5 vaccination, days in milk (DIM) at onset of CM, and herd effect as well as each 2-way interaction between the 3 factors. The adjusted daily milk for 21 d following CM was 7.6 kg greater among J5 vaccinates than controls; however, this protective effect of vaccination waned with increasing DIM at onset of CM. A mixed linear model with autoregressive order 1 [AR(1)] correlation structure estimated the daily milk production of any cow (whether or not she had CM) on a given DIM. Cows with CM caused by nonagalactiae streptococci, Staphylococcus aureus, Escherichia coli, or Klebsiella lost significant daily milk production for the entire lactation relative to nonmastitic cows. Another mixed linear model for only coliform CM cases (E. coli, Klebsiella, and Enterobacter) within the first 50 DIM showed milk loss for 21 d following coliform CM to be significantly less for J5 vaccinates than for controls, by 6 to 15 kg per day. Cows were significantly less likely to become pregnant if they had CM caused by E. coli (42% pregnant) or Streptococcus spp. (38% pregnant), whereas 78% (342/437) of cows with no mastitis conceived. Days open (number of days from calving until pregnancy) averaged 131 d for cows with no CM and 162 d for cows that had at least one case of CM. Days until conception, days until last breeding, days open, times bred, and percentage of cows pregnant by 200 DIM were not changed with J5 vaccination. Nonetheless, an important benefit of the use of J5 bacterin appears to be reduction of the loss of daily milk production following CM, whether all cases or only those caused by coliform bacteria were considered.

Cleanliness scores as indicator of Klebsiella exposure in dairy cows.

This study was designed to explore the relationship between cow and udder cleanliness scores and the risk of isolation of Klebsiella spp. from lower hind legs and teat ends, respectively. The distribution of Klebsiella species was compared among isolates from teat ends, legs, and cases of clinical mastitis obtained from 2 dairy farms in New York State, with 850 and 1,000 cows, respectively. Farms were visited twice approximately 4 wk apart in August and September 2007 to obtain cleanliness scores and swabs from legs and teats. Isolates of Klebsiella clinical mastitis from each farm were collected from July through October 2007. Two studies were conducted. In the first study, whole-cow cleanliness of a purposive sample of 200 lactating cows was scored using a 4-point scale, and swabs were taken from their lower hind legs. In the second study, udder cleanliness of a separate convenience sample of 199 lactating cows was scored in the milking parlor, and swabs were taken from their teat ends before and after premilking udder preparation. Prevalence of Klebsiella spp. on legs and teat ends before udder preparation was 59 and 60%, respectively. Logistic regression was used to explore the association between isolation of Klebsiella spp. and cleanliness scores. Cow cleanliness scores and udder cleanliness scores were not associated with detection of Klebsiella on legs and on teats before udder preparation, respectively. After udder preparation, 43% of previously Klebsiella positive teat end samples remained positive, with significant differences between farms and months. Teats from dirty udders were significantly more likely to test positive for Klebsiella after udder preparation than teats from clean udders. The proportion of Klebsiella pneumoniae and Klebsiella oxytoca isolates was similar for isolates from teat end swabs and clinical mastitis cases, supporting the notion that the presence of Klebsiella on teat ends may lead to opportunistic intramammary infections. Udder cleanliness scores could be used as a management tool to monitor the risk of exposure to Klebsiella spp. on teat ends.

Comparison of J5 vaccinates and controls for incidence, etiologic agent, clinical severity, and survival in the herd following naturally occurring cases of clinical mastitis.

Holstein dairy cattle in 3 commercial herds were randomly allocated to J5 vaccination (n = 251) or untreated control (n = 306) groups. There were 221 new cases of clinical mastitis (CM) affecting 120 cows. Coliform mastitis cases had a higher percentage of severe quarter swelling or signs of systemic illness among control cows but not among J5 vaccinates, in comparison to noncoliform cases. Culling or death from CM affected 13 controls (4.3%) and 4 vaccinates (1.6%), with losses occurring earlier in lactation among controls, a higher hazard (probability of a cow dying on each day of lactation) for controls than vaccinates. The J5 vaccination was significantly associated with protection from culling for mastitis among the 15 Klebsiella cases; 2 out of 10 (20%) Klebsiella-infected controls were culled and 0 out of 5 vaccinates were culled. Cows in second lactation were at reduced hazard of culling for mastitis compared with older animals, even when adjusting for effects of J5 vaccination. When all CM cases (including subsequent new cases during the same lactation and multiple quarters or pathogens within the same cow on the same day) were evaluated, for the 221 cases of CM, the rate was significantly higher among vaccinates than controls (0.10 and 0.07 cases/30 d in milk, respectively). This was because J5 vaccinates had more subsequent new cases of CM in the same cow than controls. Pathogens isolated, which included mainly environmental bacteria, were not different among J5 vaccinates and controls. Immunization with J5 was associated with protection against severe clinical coliform mastitis signs, culling, and death loss from CM but not with any reduction in overall CM.

Morphological characterization of spinal cord dorsal horn lamina I neurons projecting to the parabrachial nucleus in the rat.

Many Rexed's lamina I neurons are nociceptive and project to the brain. Lamina I projection neurons can be classified as multipolar, fusiform, or pyramidal, based on cell body shape and characteristics of their proximal dendrites in the horizontal plane. There is also evidence that both multipolar and fusiform cells are nociceptive and pyramidal neurons nonnociceptive. In this investigation we identified which types of lamina I neurons belong to the spinoparabrachial tract in the rat and characterized them regarding the presence or absence of neurokinin-1 receptor (NK-1r) immunoreactivity. For this, cholera toxin subunit B (CTb), conjugated to a fluorescent marker was injected unilaterally into the parabrachial nucleus. Sections were additionally stained for the detection of NK-1r immunoreactivity and were examined using fluorescence and confocal microscopy. Serial confocal optical sections and 3D reconstructions were obtained for a considerable number of neurons per animal. Using immunofluorescence, we assessed the proportion of lamina I neurons belonging to the spinoparabrachial (SPB) tract and/or expressing NK-1r. The relative distribution of neurons belonging to the SPB tract was: 38.7% multipolar, 36.8% fusiform, 22.7% pyramidal, and 1.9% unclassified. Most of the SPB neurons expressing NK-1r were either multipolar or fusiform. Pyramidal SPB neurons were seldom immunoreactive for NK-1r, an observation that provides further support to the concept that most lamina I projection neurons of the pyramidal type are nonnociceptive. In addition, our study provides further evidence that these distinct morphological types of neurons differ in their phenotypic properties, but not in their projection patterns.

Chemotherapy-evoked painful peripheral neuropathy: analgesic effects of gabapentin and effects on expression of the alpha-2-delta type-1 calcium channel subunit.

Chemotherapeutics in the taxane and vinca-alkaloid classes sometimes produce a painful peripheral neuropathy for which there is no validated treatment. Experiments with rat models of paclitaxel- and vincristine-evoked pain suggest that these conditions may not respond to all of the analgesics that have efficacy in other models of painful peripheral neuropathy. We tested gabapentin as a potential analgesic for paclitaxel- and vincristine-evoked pain. We used a repeated dosing paradigm because there are precedents showing that repeated drug exposure may be necessary to demonstrate analgesia in neuropathic pain models. Gabapentin is believed to work via binding to voltage-gated calcium channels that contain the alpha-2-delta type-1 (alpha(2)delta-1) subunit, and the expression of this subunit is known to be increased in some painful peripheral neuropathy models. Thus we also examined whether the paclitaxel-evoked pain syndrome was accompanied by an alpha(2)delta-1 increase, and whether gabapentin had any effect on subunit expression. We found that the paclitaxel- and vincristine-evoked mechano-allodynia and mechano-hyperalgesia were significantly reduced by gabapentin, but only with repeated dosing. Paclitaxel-evoked painful peripheral neuropathy was associated with an increased expression of the alpha(2)delta-1 subunit in the spinal dorsal horn, but not in the dorsal root ganglia. The spinal cord increase was normalized by repeated gabapentin injections. Together, these findings suggest that repeated dosing with gabapentin may be beneficial in patients with chemotherapy-evoked painful peripheral neuropathy and that gabapentin's mechanisms of action may include normalization of the nerve injury-evoked increase in calcium channel alpha(2)delta-1 subunit expression.

Autonomic fibre sprouting and changes in nociceptive sensory innervation in the rat lower lip skin following chronic constriction injury.

In this study we used immunocytochemistry to investigate whether autonomic fibres sprouted in the skin of the lower lip in a rat model of neuropathic pain. We used a bilateral chronic constriction injury (CCI) of the mental nerve (MN), a branch of the trigeminal nerve. In this model, we also studied the accompanying changes in peptidergic [calcitonin gene-related peptide (CGRP)-immunoreactive] sensory fibres, as well as in trkA receptor immunoreactivity in the sensory nerves. Autonomic (sympathetic and parasympathetic) fibre sprouting was first observed 1 week post-injury with a peak in the number of sprouted fibres occurring at 4 and 6 weeks post-CCI. CGRP-IR fibres almost disappeared at 2 weeks post-CCI, but quickly sprouted, leading to a significant peak above sham levels 4 weeks post-injury. trkA receptor expression was found to be up-regulated in small cutaneous nerves 4 weeks post-CCI, returning to sham levels by 8 weeks post-CCI. There was no sympathetic fibre sprouting in the trigeminal ganglion following CCI. At 4 weeks post-CCI, rats displayed spontaneous, directed grooming to the area innervated by the MN that was not seen in sham animals, which we interpreted as a sign of spontaneous pain or dysesthesiae. Collectively, our findings indicate that as a result of autonomic sprouting due to CCI of the MN, remaining intact nociceptive fibres may potentially develop sensitivity to sympathetic and parasympathetic stimulation, which may have a role in the generation of abnormal pain following nerve injury.

Effect of clinical mastitis on the lactation curve: a mixed model estimation using daily milk weights.

The objective of this study was to estimate the milk production losses associated with clinical mastitis using mixed linear models and correlation structures that have not been available previously. Data used included computer-recorded daily milk yields and detailed and accurate recordings of clinical mastitis cases. Two commercial Holstein dairy farms in New York State participated in the study, one with 650 lactating cows and another that began the study with 830 lactating cows and increased to 1120 cows by the end of the study. Cows on both farms were housed in free stall barns and milked 3 times daily in milking parlors. Electrical conductivity was used as a diagnostic aid for clinical mastitis on both farms. Date of clinical onset was recorded for every episode of clinical mastitis as well as for 8 other diseases defined using standardized case definitions (dystocia, milk fever, retained placenta, metritis, ketosis, displaced abomasum, lameness, and cystic ovarian disease) during the study period of October 1, 1999 to July 31, 2001. The mixed linear model for explaining variation in the outcome variable daily milk yield relative to non-mastitic herdmates found the terms for all 9 diseases studied, including clinical mastitis, significant. The model with an autoregressive correlation structure was preferred based on -2 * log likelihood, Akaike's information criterion, and Bayesian information criterion as well as savings in degrees of freedom. Separate analyses were run for first lactation cows and for second-plus lactation cows because their lactation curves were shaped differently. Adjusting for the effects of the other 8 diseases, milk production loss from clinical mastitis during the whole lactation was estimated as approximately 598 kg for second-plus lactation cows. However, cows that contracted mastitis had a daily production advantage of 2.6 kg over their herdmates until they contracted the disease. When compared with this potentially higher milk production, the total loss from clinical mastitis was estimated as 1181 kg.

Use of a craniofacial miniplate for internal fixation in a young child with cervical instability. Case report.

Commercially available anterior cervical internal fixation devices are designed for placement in adults and older children. Use of these systems in preschool-aged children is precluded due to the small size of their cervical vertebral bodies (VBs). The authors describe a 2-year-old boy who suffered a C3-4 injury, resulting in complete ligamentous disruption. Because of the gross cervical instability, they elected to perform surgery via posterior and anterior approaches, supplemented with internal fixation, during the same operation. The purpose of the anterior internal fixation device is to deliver compressive forces onto the interbody graft and keep it in place, thus optimizing the potential for a successful fusion. Because of the discrepancy in size between the VBs and the plate and screws, however, the authors were unable to use any of the standard anterior cervical fixation devices. Instead, they implanted a craniofacial miniplate, and the patient was required to wear an external halo brace. The miniplate provided enough stability to allow for a solid fusion. The authors believe that this technique is a reasonable option in young children who require anterior cervical fixation.