In vitro biosynthesis of plasma proteins under ischemic conditions of closed-circuit perfusion of healthy and intoxicated rabbit liver

We are elaborating on the kinetics and mechanisms of septic rabbit liver to de novo biosynthesize acute-phase response (APR) proteins under in vitro conditions of deepening ischemia in reference to their in vivo prevalence in serum and cerebrospinal fluids (CSF) collected at predetermined times. The significance of the data is interpreted as relevant to grafting cadaveric liver into end-stage liver diseased patients and APR-induced ischemic heart diseases (IHD). Hepatic APR was induced by CCl(4)-intubation, and the administration of cholera toxin (CT) or scorpion venom (SV), or both, to rabbits. Hepatic functional efficiency, in terms of biosynthesis of APR proteins in closed circuit perfusion of the isolated intoxicated liver with oxygenated saline or L-15 media paralleled the two-dimensional immunoelectrophoresis (2D-IEP) spectrum of APR serum proteins at time of liver isolation. We are suggesting: (a) in vitro biosynthesis of plasma proteins by isolated perfused liver is the result of in vivo decoded and retained APR inflammatory signals; and (b) decoded inflammatory signals are expressed not withstanding the perfusate's organic composition. Furthermore, 90 min of ischemic perfusion in saline or L-15 medium precipitated mitochondrial aberrations which resulted in further deterioration of de novo biosynthesis of APR plasma proteins. Regardless of the nature of the inflammatory stimuli, mitochondrial aberrations rendered the perfused organ a biologically inert tissue mass that was incapable of resuming biological function upon perfusion with oxygenated L-15 medium. This is most likely due to ischemia-induced irreversible hepatic necrosis. Thus, in vitro aberrations of mitochondrial function(s) critically limit the capability of the isolated liver to resume its organic function to sustain biosynthesis of de novo plasma proteins. Extrapolation of these results to the surgical management of end-stage liver diseases points to the importance of the status and the handling protocol(s) of the cadaver donor liver prior to successful grafting. We conclude that although histology of a cadaver liver may reveal well-preserved hepatic cellular organelles with at least minimal intra- and intercellular communication required for viable hepatic function, we deem it essential to further define acceptable minimal capabilities to de novo biosynthesize plasma proteins by a cadaver liver as a measure of its functional viability and suitability for transplantation. Ultimately, this measure may improve the success of liver transplants with minimal surgical and drug interventions.     

Respiratory effects of buprenorphine/naloxone alone and in combination with diazepam in naive and tolerant rats

Respiratory depression has been attributed to buprenorphine (BUP) misuse or combination with benzodiazepines. BUP/naloxone (NLX) has been marketed as maintenance treatment, aiming at preventing opiate addicts from self-injecting crushed pills. However, to date, BUP/NLX benefits in comparison to BUP alone remain debated. We investigated the plethysmography effects of BUP/NLX in comparison to BUP/solvent administered by intravenous route in naive and BUP-tolerant Sprague-Dawley rats, and in combination with diazepam (DZP) or its solvent. In naive rats, BUP/NLX in comparison to BUP significantly increased respiratory frequency (f, P < 0.05) without altering minute volume (V_E). In combination to DZP, BUP/NLX significantly increased expiratory time (P < 0.01) and decreased f (P < 0.01), tidal volume (V_T, P < 0.001), and V_E (P < 0.001) while BUP only decreased V_T (P < 0.5). In BUP-tolerant rats, no significant differences in respiratory effects were observed between BUP/NLX and BUP. In contrast, in combination to DZP, BUP/NLX did not significantly alter the plethysmography parameters, while BUP increased inspiratory time (P < 0.001) and decreased f (P < 0.01) and V_E (P < 0.001). In conclusion, differences in respiratory effects between BUP/NLX and BUP are only significant in combination with DZP, with increased depression in naive rats but reduced depression in BUP-tolerant rats. However, BUP/NLX benefits in humans remain to be determined.     

Peritoneal Absorption of Cefoperazone in Rats

The peritoneal absorption of cefoperazone, administered by intraperitoneal (ip) perfusion in a large volume (100mg/40ml), was investigated in rats. Its pharmacokinetics was also studied after ip or intravenous (iv) injection of 100mg/kg in two groups of rats. The peritoneal uptake after the two modes of ip administration was rapid, peaking in less than 20 minutes and the means of peak concentrations were similar. The peak remained high in the group perfused with a large volume for at least 4 hours, which was the end of sample collection. In addition, the absorption half-life and the fraction (F) reaching systemic circulation were calculated and found to be 10.0±2.5 min and 0.93, respectively. A brief distribution phase (t½α8.0 ± 0.67 minutes) appeared only after the iv bolus. Otherwise the decline in serum concentration was monoexponential with half-lives of 39.0 ± 4.0 and 63.6 ± 7.5 min for the iv and ip injected groups, respectively.

The stability of cefoperazone in plasma was also investigated in this study. It was found to be unstable at physiological pH even at - 30° C and the samples collected should be buffered in acidic media to optimize stability. The degradation process is likely to contribute to its elmimination kinetics during in vivo administration.     

Effects of Epigallocatechin-3-gallate on lead-induced oxidative damage

Recent studies have shown that lead (Pb) could disrupt the prooxidant/antioxidant balance of tissue which leads to biochemical and physiological dysfunction. Epigallocatechin-3-gallate (EGCG), a catechin polyphenols component, is found to be an effective antioxidant. The present study investigated whether EGCG administration could reverse the changes on redox states in rat hippocampus caused by lead exposure. The association between redox status changes and long-term potentiation (LTP) in CA1 area of hippocampus were also examined. Wistar rats exposed to lead from postnatal day 1 were followed by 10 days of EGCG (10, 25 and 50 mg/kg) administration through intraperitoneally (ip), and the rats were sacrificed for experiments at the age of 21–23 days. The experimental results showed that glutathione (GSH) and superoxide dismutase (SOD) activity decreased accompanied with LTP amplitude decrease in CA1 area of hippocampus in the lead-exposed group. EGCG supplementation following lead intoxication resulted in increases in the GSH and SOD levels and increases in the LTP amplitude. Malondialdehyde (MDA) levels, a major lipid peroxidation byproduct, increased following lead exposure and decreased following EGCG treatment. In hippocampal neuron culture model, lead exposure (20 μM) significantly inhibited the viability of neurons which was followed by an accumulation of ROS and a decrease of mitochondrial membrane potential (ΔΨ_m). Treatment by EGCG (10–50 μM) effectively increased cell viability, decreased ROS formation and improved ΔΨ_m in hippocampal neurons exposed to lead. These observations suggest that EGCG is a potential complementary agent in the treatment of chronic lead intoxication through its antioxidative character.     

嗜肺军团菌ip基因的克隆及其原核表达

目的克隆嗜肺军团菌主要免疫原蛋白ip基因,构建重组质粒pET-ip,并在原核系统中表达。方法采用聚合酶链反应(PCR),从嗜肺军团菌基因组DNA中扩增军团菌主要免疫原蛋白ip基因,并将其定向克隆至原核表达载体pET-32a( ),构建原核表达重组质粒pET-ip,经限制性内切酶鉴定、PCR及测序分析后,转化宿主菌大肠杆菌BL21,IPTG诱导表达,产物进行SDS-PAGE电泳、免疫印迹分析鉴定。结果扩增出了792 bp完整的ip基因,构建的原核表达重组质粒pET-ip表达出49 kDa Trx-IP的融合蛋白质,Western-blot证实获得蛋白为所需要的目的蛋白。结论成功构建了军团菌ip基因的原核表达载体,并在大肠杆菌中得到了高效表达。     

Functional subsets of neurons in somatosensory thalamus of the cat

Functional properties of somatosensory thalamic neurons isolated within a region encompassing nucleus ventralis posterolateralis were studied in chloralose-anesthetized cats. A sample of these neurons responding to electrical stimulation of the contralateral forepaw was reconstructed by pooling data from 46 individual animals on the basis of unit spatial location. Seventy percent of the 640 neurons studied responded only to the contralateral forepaw; they had small receptive fields as determined by natural stimulation, generally on the digits and the dorsum of the paw, and 75% of this group were excited by light touch or deflection of hairs. Thirty percent of the neurons studied responded to the contralateral forepaw and to other limbs as well, their larger receptive fields often being bilateral. Approximately 50% of these wide-field neurons were excited by gentle mechanical stimulation. Some cells of this type were identified as projection neurons by virtue of their antidromic activation from the S I forepaw area of cerebral cortex. The wide-field neurons described here are not adequately explained as vestigial aberrations persisting at the feline stage in an evolutionary trend toward strictly somatotopic thalamic representation. Comparison of the dynamic response properties (latency, spikes per discharge, and frequency-following) of small- and wide-field somatosensory thalamic neurons suggests that they are activated via different input mechanisms and that each has a particular functional role in somesthesis.     

Wide-field conditioning effects on small-field neurons in the posterior sigmoid gyrus of domestic cats

Single neurons were isolated in four different recording sites in sensorimotor cortex of the domestic cat, all sites being topographically related to the contralateral forepaw. Conditioning-testing interactions were made on 409 small-field neurons, the testing input being the contralateral forepaw (also the ipsilateral forepaw in the case of bilateral-field neurons) and the conditioning input being any paw that was ineffective in exciting the neuron. About 29% of the neurons showed inhibitory interactions and 23% showed facilitatory interactions. Of these, about 5.5% showed both effects, according to the conditioning site. Nearly 54% of the neurons failed to show any interaction effects. Most of the latter were isolated in the two posterior recording sites, located in somatosensory area I; the interaction effects were found predominantly in the two anterior recording sites, located in agranular tissue. Neurons that responded only to contralateral forepaw stimulation (sa neurons) and showed facilitatory interactions from the other three paws had response properties characteristic of neurons that respond to all four paws (m neurons). The sa neurons that showed inhibitory interactions from the other three paws did not differ significantly in response properties from those that showed no interaction effects. These findings are relevant to current criteria for the classification of sensorimotor cortex neurons and lead to remarks on the possible thalamic routes that mediate the interaction effects.     

一种制备急性CO中毒迟发性脑病动物模型的新方法

目的 建立一种新方法制备的一氧化碳中毒迟发性脑病的动物模型,以便深入探索迟发性脑病的发病机制。方法 将家兔随机分成单次染毒组和连续染毒组,采用腹腔注射CO法染毒;单次染毒组按200ml/kg剂量注射1次,连续染毒组按6h间隔追加注射3次,监测染毒后24h内各组家兔血液HbCO、SOD和MDA的变化,并在不同时间处死动物后采用病理学技术鉴定脑组织损伤情况。结果 腹腔CO染毒后,家兔迅速出现明显的中毒症状,血HbCO水平显著增高,1—2h达高峰,6h后呈现下降趋势,单次染毒组可在14h后将CO完全排出体外,连续染毒组HbCO水平则可维持在45％以上达24h;染毒家兔SOD活力明显下降,MDA含量显著升高。病理学检查显示,染毒急性期家兔脑组织损伤严重,并在随后的3—21d内出现继发性的脑组织坏死,海马及皮层尤为显著。结论 采用腹腔连续间隔注射CO可成功建立急性一氧化碳中毒迟发性脑病动物模型,从而为进一步探讨其发病机制奠定了工作基础。     

Pre-ischemic treatment with memantine reversed the neurochemical and behavioural parameters but not energy metabolites in middle cerebral artery occluded rats

In the present study, Memantine (MN) an uncompetitive N-methyl-d-aspartate (NMDA) open channel blocker has been investigated for its suitable therapeutic time-window on the basis of its influence on behavioural and biochemical changes in rats subjected to transient focal ischemia. MN (20 mg/kg, ip) was administered at pre, during and post ischemic state and the extent of neuroprotection was compared to ascertain its therapeutic time-window in stroke treatment. Neuroprotective effect was assessed by measuring glutamate, glutamine synthetase, glutathione, Na⁺K⁺ATPase, adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide (NAD), lactate and pyruvate levels. Middle cerebral artery occlusion produced neurological deficits, anxiogenic behaviour, histological changes, increased glutamate levels along with depletion of Na⁺K⁺ATPase, energy stores such as ATP, NAD, lactate, and antioxidant glutathione. MN significantly restored glutamate, glutamine synthetase, Na⁺K⁺ATPase and lactate levels on preischemic administration. In addition, MN reversed the altered neurological and behavioural paradigms significantly and prevented the neurodegeneration on preischemic treatment. However, it failed to exert any effect on energy metabolite (ATP and NAD) levels irrespective of the treatment phase. Based on the present data, it is summarized that the suitable therapeutic time window of MN is preischemic phase in stroke and it possesses only a subjective role in reversing ischemic brain biochemical alterations preferentially in favor of neuronal homeostasis.     

Quantitative determination of the biological activity of botulinum toxin type A by measuring the compound muscle action potential (CMAP) in rats

Quantitative determination of the biological activity of botulinum toxin type A usually depends on the LD₅₀ method after intraperitoneal injection into mice. This method requires a large number of mice to determine the toxic activity at a high level of precision and 3–4 days to obtain the results. Techniques to replace the LD₅₀ method have been attempted at various institutes. As a substitute for this method, by directly measuring the inhibition of neuromuscular transmission after the administration of a toxin, a method to quantitatively assess the toxin's activity by determining the compound muscle action potential (CMAP) was examined.Toxin solutions were injected into the rat gastrocnemius muscle, and that of the CMAP amplitude was determined over time. The CMAP amplitude decreased over 4 days after the injection of the toxin, and then slowly recovered. A dose-response relationship was noted for each dose, and a linear relation was observed between 0.01 and 30 U on the 1st day. From these results, we propose the CMAP as a substitute for the LD₅₀ method to examine the activity of toxin products as it is simple and reliable, reduces the number of experimental animals required, and lowers pain levels.