猪繁殖与呼吸综合征病毒感染肺泡巨噬细胞后差异表达膜蛋白的鉴定与分析

猪繁殖与呼吸综合征(porcine reproductive and respiratory syndrome,PRRS)是危害养猪业发展的重要传染病之一。2006年我国首次爆发高致病性PRRS(highly-pathogenicPRRS,HP-PRRS),与经典的PRRS相比,HP-PRRS发病率和死亡率均显著升高。目前,对高致病性猪繁殖与呼吸综合征病毒(highly-pathogenic porcine reproductive and respiratory syndrome virus,HP-PRRSV)引起的高发病率和高死亡率的机制仍不清楚。本研究利用1MOI的EGFP标记的HP-PRRSV毒株HuN4(rHuN4-EGFP)和其体外传代致弱毒株HuN4-F112(rHuN4-F112-EGFP)分别感染肺泡巨噬细胞(pulmonaryalveolarmacrophage,PAM),24h后通过流式细胞术,从FITC通道收集被PRRSV强弱毒感染的PAM,提取膜蛋白进行Shotgun质谱分析。试验最终获得82个在强弱毒感染PAM过程中存在差异表达的膜蛋白。感染rHuN4-EGFP的样品与未感染样品相比,有72个蛋白特异表达;感染rHuN4-F112-EGFP的样品与未感染样品相比,有12个蛋白特异表达。通过GO分析发现,这些蛋白主要参与代谢、生物调节与细胞加工过程,大多数蛋白具有结合、催化活性。进一步在MARC-145细胞上过表达部分差异膜蛋白,发现其中前蛋白转化酶枯草杆菌蛋白酶9(proprotein coverteases subtilism/kexin 9,PCSK9)能明显抑制PRRSV强毒株和弱毒株的复制,Clusterin、Apoliprotein C-II能促进PRRSV强毒株复制。本研究表明利用Shotgun质谱技术鉴定出的差异表达膜蛋白对PRRSV复制有影响,深入分析PRRSV感染后差异表达的膜蛋白变化将有利于进一步明确其致病机制。     

Determination of the minimum alveolar concentration (MAC) and cardiopulmonary effects of sevoflurane in sheep

Objective

To determine sevoflurane’s minimum alveolar concentration (MAC_SEVO) and its cardiopulmonary effects in sheep.

An alveolar recruitment maneuver followed by positive end-expiratory pressure improves lung function in healthy dogs undergoing laparoscopy

Objective

To evaluate the effects of an alveolar recruitment maneuver (ARM) followed by 5 cmH₂O positive end-expiratory pressure (PEEP) in dogs undergoing laparoscopy.

RADIOGRAPHIC BONE LOSS CORRELATIONS AND TECHNETIUM-99m-MDP BONE UPTAKE IN LIGATURE-INDUCED PERIODONTAL DISEASE IN THE BEAGLE

The goal of this study was to develop an animal model for the study of acute periodontal disease using silk ligatures in combination with a soft diet in eleven purebred beagle dogs. The silk ligatures were placed subgingivally on the mandible second and fourth premolar on one side of the mouth; the opposite side served as a control. Dogs were monitored during the 16–20 weeks of ligature placement, and for 48 weeks after ligature removal. Development of periodontal disease was evaluated by radiopharmaceutical uptake into bone, radiographic evidence of alveolar bone loss, attachment loss, gingival index and prostaglandin level. Bone loss occurred on the ligatured side during the ligature phase of the study. Radiopharmaceutical uptake was correlated with radiographic evidence of bone loss during the ligature phase. No significant bone loss occurred during the post-ligature phase. Progressive periodontal disease was induced during ligature placement. However, a chronic less aggressive form was not sustained by soft diet alone after ligature removal.     

The effect of midazolam on the end-tidal concentration of isoflurane necessary to prevent movement in dogs

ObjectiveTo determine the possible additive effect of midazolam, a GABA_A agonist, on the end-tidal concentration of isoflurane that prevents movement (MAC_NM) in response to noxious stimulation.Study designRandomized cross-over experimental study.AnimalsSix healthy, adult intact male, mixed-breed dogs.MethodsAfter baseline isoflurane MAC_NM (MAC_NM-B) determination, midazolam was administered as a low (LDS), medium (MDS) or high (HDS) dose series of midazolam. Each series consisted of two dose levels, low and high. The LDS was a loading dose (Ld) of 0.2 mg kg^?1 and constant rate infusion (CRI) (2.5 μg kg^?1 minute^?1) (LDL), followed by an Ld (0.4 mg kg^?1) and CRI (5 μg kg^?1 minute^?1) (LDH). The MDS was an Ld (0.8 mg kg^?1) and CRI (10 μg kg^?1 minute^?1) (MDL) followed by an Ld (1.6 mg kg^?1) and CRI (20 μg kg^?1 minute^?1) (MDH). The HDS was an Ld (3.2 mg kg^?1) and CRI (40 μg kg^?1 minute^?1) (HDL) followed by an Ld (6.4 mg kg^?1) and CRI (80 μg kg^?1 minute^?1) (HDH). MAC_NM was re-determined after each dose in each series (MAC_NM-T).ResultsThe median MAC_NM-B was 1.42. MAC_NM-B did not differ among groups (p >0.05). Percentage reduction in MAC_NM was significantly less in the LDS (11 ± 5%) compared with MDS (30 ± 5%) and HDS (32 ± 5%). There was a weak correlation between the plasma midazolam concentration and percentage MAC_NM reduction (r = 0.36).Conclusion and clinical relevanceMidazolam doses in the range of 10–80 μg kg^?1 minute^?1 significantly reduced the isoflurane MAC_NM. However, doses greater than 10 μg kg^?1 minute^?1 did not further decrease MAC_NM indicating a ceiling effect.     

Effect of sevoflurane concentration on visual evoked potentials with pattern stimulation in dogs

The purpose of this study was to investigate the effects of sevoflurane concentration on canine visual evoked potentials with pattern stimulation (P-VEPs). Six clinically normal laboratory-beagle dogs were used. The minimum alveolar concentration (MAC) of sevoflurane was detected from all subjects by tail clamp method. The refractive power of the right eyes of all subjects was corrected to −2 diopters after skiascopy. For P-VEP recording, the recording and reference electrode were positioned at inion and nasion, respectively, and the earth electrode was positioned on the inner surface. To grasp the state of CNS suppression objectively, the bispectral index (BIS) value was used. The stimulus pattern size and distance for VEP recording were constant, 50.3 arc-min and 50 cm, respectively. P-VEPs and BIS values were recorded under sevoflurane in oxygen inhalational anesthesia at 0.5, 1.0, 1.5, 2.0, 2.5 and 2.75 sevoflurane MAC. For analysis of P-VEP, the P100 implicit time and N75-P100 amplitude were estimated. P-VEPs were detected at 0.5 to 1.5 MAC in all dogs, and disappeared at 2.0 MAC in four dogs and at 2.5 and 2.75 MAC in one dog each. The BIS value decreased with increasing sevoflurane MAC, and burst suppression began to appear from 1.5 MAC. There was no significant change in P100 implicit time and N75-P100 amplitude with any concentration of sevoflurane. At concentrations around 1.5 MAC, which are used routinely to immobilize dogs, sevoflurane showed no effect on P-VEP.     

The effect of experimentally induced hypothyroidism on the isoflurane minimum alveolar concentration in dogs

ObjectiveTo determine the effect of experimentally induced hypothyroidism on isoflurane (ISO) minimum alveolar concentration (MAC) in dogs.Study designProspective experimental study.AnimalsEighteen adult female mongrel dogs, age 2–4 years and weighing 8.2–13.1 kg.MethodsHypothyroidism was induced in nine dogs by the intravenous administration of 1 mCi kg⁻¹ of ¹³¹Iodine. The remaining nine dogs served as controls. Dogs were studied 9–12 months after the induction of hypothyroidism. Anesthesia was induced with ISO in oxygen via a mask. The trachea was intubated, and anesthesia was maintained using ISO in oxygen using a semi-closed rebreathing circle system. The dogs were mechanically ventilated to maintain an end-tidal carbon dioxide concentration between 35 and 45 mmHg. End-tidal ISO concentrations were measured with an infrared gas analyzer. The MAC was determined in duplicate using a tail clamp technique. The mean values for the groups were compared using a two sample t-test.ResultsThe mean ± SD MAC of isoflurane in the hypothyroid and euthyroid dogs was 0.98 ± 0.31% and 1.11 ± 0.26%, respectively. The mean MAC of isoflurane in hypothyroid dogs was not significantly different from the mean MAC of isoflurane in the control dogs (p=0.3553).Conclusion and clinical relevanceThe MAC of ISO in dogs was not significantly affected by experimentally induced hypothyroidism. The dose of ISO in dogs with hypothyroidism does not need to be altered.     

GABA(A) receptor antagonism increases NMDA receptor inhibition by isoflurane at a minimum alveolar concentration

ObjectiveAt the minimum alveolar concentration (MAC), isoflurane potentiates GABA_A receptor currents and inhibits NMDA receptor currents, and these actions may be important for producing anesthesia. However, isoflurane modulates GABA_A receptors more potently than NMDA receptors. The objective of this study was to test whether isoflurane would function as a more potent NMDA receptor antagonist if its efficacy at GABA_A receptors was decreased.Study designProspective experimental study.AnimalsFourteen 10-week-old male Sprague–Dawley rats weighing 269 ± 12 g.MethodsIndwelling lumbar subarachnoid catheters were surgically placed in isoflurane-anesthetized rats. Two days later, the rats were anesthetized with isoflurane, and artificial CSF containing either 0 or 1 mg kg^?1 picrotoxin, a GABA_A receptor antagonist, was infused intrathecally at 1 μL minute^?1. The baseline isoflurane MAC was then determined using a standard tail clamp technique. MK801 (dizocilpine), an NMDA receptor antagonist, was then administered intravenously at 0.5 mg kg^?1. Isoflurane MAC was re-measured.ResultsPicrotoxin increased isoflurane MAC by 16% compared to controls. MK801 significantly decreased isoflurane MAC by 0.72% of an atmosphere in controls versus 0.47% of an atmosphere in rats receiving intrathecal picrotoxin.Conclusions and clinical relevanceA smaller MK801 MAC-sparing effect in the picrotoxin group is consistent with greater NMDA antagonism by isoflurane in these animals, since it suggests that fewer NMDA receptors are available upon which MK801 could act to decrease isoflurane MAC. Decreasing isoflurane GABA_A potentiation increases isoflurane NMDA antagonism at MAC. Hence, the magnitude of an anesthetic effect on a given channel or receptor at MAC may depend upon effects at other receptors.