A novel large animal model of recurrent migraine established by repeated administration of inflammatory soup into the dura mater of the rhesus monkey.

Several animal models of migraine have been established, and those based on trigeminovascular system activation are widely accepted. However, most of these models have been established on lower animals, such as rodents, and involve only a single administration of a noxious stimulus. In this study, an inflammatory soup (10 µL), consisting of prostaglandin E2 (0.2 mM), serotonin (2 mM), bradykinin (2 mM) and histamine (2 mM), was injected into the dura mater of conscious rhesus monkeys through an indwelling catheter. The infusion started on day 8 and was repeated every 3 days, for a total of six administrations, to induce neurogenic inflammation. We performed behavioral assessments and measured the expression of the oncogene c-fos, neuronal nitric oxide synthase (nNOS) and calcitonin gene related peptide (CGRP) in the trigeminal system and in multiple brain regions involved in pain processing by immunohistochemical staining. Compared with monkeys in the control group, three of the four animals in the inflammatory soup group displayed decreased motor behaviors, and two showed increased ipsilateral nose and mouth secretions during the stimulus period. Higher expression levels of c-fos, nNOS and CGRP were found in various brain areas of experimental animals compared with controls, including the trigeminal nucleus caudalis, thalamus, hypothalamus, midbrain, pons and other areas involved in pain perception. These results suggest that repeated inflammatory soup stimulation of the dura activates the trigeminovascular system and produces migraine-like pathological changes and abnormal behaviors in conscious rhesus monkeys.

Assessment of a passive immunity mouse model to quantitatively analyze the impact of neutralizing antibodies on adeno-associated virus-mediated gene transfer.

Adeno-associated viruses (AAVs) are common infective agents of primates. As such, healthy primates carry a large pool of AAV-specific neutralizing antibodies (NAbs), which inhibit AAV-mediated gene transfer therapeutic strategies. Thus, a clinical method to screen patient candidates for AAV-specific NAbs prior to treatment, especially with the frequently used AAV8 capsid component, will facilitate individualized treatment design and enhance therapeutic efficacy. In this study, we evaluated the efficacy and sensitivity of a passive immunity mouse model to quantitatively assess anti-AAV8 NAb titers, as compared to an in vitro immunoassay. The passive transfer model was established in C57BL/6 mice by tail vein injection of pre-defined sera from 23 male rhesus monkeys. The mice were then administered low dose (3e10 GC/mouse) self-complementary (sc) AAV8. The in vitro NAb assay indicated that 69.57% of the rhesus donors had pre-existing anti-AAV8 NAb. The in vivo NAb assay, however, was better able to detect low NAb titer (≤ 1:5), which can mediate neutralization in vivo. Indeed, 17 rhesus donors (74.0%) had pre-existing anti-AAV8 neutralization by in vivo NAb assay. Our findings indicated that the in vivo NAb assay is superior to the in vitro assay for detecting low NAb titers.

(1) H-nuclear magnetic resonance-based metabonomic analysis of brain in rhesus monkeys with morphine treatment and withdrawal intervention.

Comprehensive cerebral metabolites involved in morphine dependence have not been well explored. To gain a better understanding of morphine dependence and withdrawal therapy in a model highly related to humans, metabolic changes in brain hippocampus and prefrontal cortex (PFC) of rhesus monkeys were measured by (1) H-nuclear magnetic resonance spectroscopy, coupled with partial least squares and orthogonal signal correction analysis. The results showed that concentrations of myoinositol (M-Ins) and taurine were significantly reduced, whereas lactic acid was increased in hippocampus and PFC of morphine-dependent monkeys. Phosphocholine and creatine increased in PFC but decreased in hippocampus after chronic treatment of morphine. Moreover, N-acetyl aspartate (NAA), γ-aminobutyric acid, glutamate, glutathione, methionine, and homocysteic acid also changed in these brain regions. These results suggest that chronic morphine exposure causes profound disturbances of neurotransmitters, membrane, and energy metabolism in the brain. Notably, morphine-induced dysregulations in NAA, creatine, lactic acid, taurine, M-Ins, and phosphocholine were clearly reversed after intervention with methadone or clonidine. Our study highlights the potential of metabolic profiling to enhance our understanding of metabolite alteration and neurobiological actions associated with morphine addiction and withdrawal therapy in primates.

Proteomic analysis of the nucleus accumbens in rhesus monkeys of morphine dependence and withdrawal intervention.

It has been known that the reinforcing effects and long-term consequences of morphine are closely associated with nucleus accumbens (NAc) in the brain, a key region of the mesolimbic dopamine pathway. However, the proteins involved in neuroadaptive processes and withdrawal symptom in primates of morphine dependence have not been well explored. In the present study, we performed proteomes in the NAc of rhesus monkeys of morphine dependence and withdrawal intervention with clonidine or methadone. Two-dimensional electrophoresis was used to compare changes in cytosolic protein abundance in the NAc. We found a total of 46 proteins differentially expressed, which were further identified by mass spectrometry analysis. The identified proteins can be classified into 6 classes: metabolism and mitochondrial function, synaptic transmission, cytoskeletal proteins, oxidative stress, signal transduction and protein synthesis and degradation. Importantly, we discovered 14 proteins were significantly but similarly altered after withdrawal therapy with clonidine or methadone, revealing potential pharmacological strategies or targets for the treatment of morphine addiction. Our study provides a comprehensive understanding of the neuropathophysiology associated with morphine addiction and withdrawal therapy in primate, which is helpful for the development of opiate withdrawal pharmacotherapies.

Transendocardial delivery of AAV6 results in highly efficient and global cardiac gene transfer in rhesus macaques.

Heart disease is the leading cause of morbidity and mortality, and cardiac gene transfer has potential as a novel therapeutic approach. We previously demonstrated safe and efficient gene transfer to the canine heart using a percutaneous transendocardial injection procedure to deliver self-complementary (sc) adeno-associated virus 6 (AAV6) vector. In the present study, we proceed with our vertical translation study to evaluate cardiac gene transfer in nonhuman primates (NHPs). We screened approximately 30 adult male rhesus macaques for the presence of neutralizing antibodies against AAV6, AAV8, and AAV9, and then selected seven monkeys whose antibody titers against these three serotypes were lower than 1/5. The animals were then randomized to receive either scAAV6 (n=3), scAAV8 (n=1), or scAAV9 (n=3) vector expressing the enhanced green fluorescent protein (EGFP) reporter gene at a dose of 5.4×10(12) genome copies/kg, which was administered according to a modified version of our previously developed transendocardial injection procedure. One animal treated with scAAV6 died secondary to esophageal intubation. The remaining animals were euthanized 7 days after gene transfer, at which time tissue was collected for analysis of EGFP expression, histopathology, and biodistribution of the vector genome. We found that (i) transendocardial delivery of AAV is safe in the NHP, (ii) AAV6 and AAV8 provide efficient cardiac gene transfer at similar levels and are superior to AAV9, and (iii) AAV6 is more cardiac-specific than AAV8 and AAV9. The results of this NHP study may help guide the development AAV vectors for the treatment of cardiovascular disease in humans.

Diffusion tensor MRI for the assessment of cerebral ischemia/reperfusion injury in the penumbra of non-human primate stroke model.

OBJECTIVES: The purpose of this study is to use diffusion tensor imaging (DTI) parameters to evaluate cerebral ischemia/reperfusion injury in the infarct core (IC) and ischemic penumbra (IP) in a rhesus transient middle cerebral artery occlusion (MCAO) model. METHODS: Seven rhesus monkeys were used to construct the MCAO model. The temporal evolution of the relative apparent diffusion coefficient (rADC) and the relative fractional anisotropy (rFA) in the IC area, infarct growth area (IG), and reversible penumbra area (RP) were investigated. RESULTS: The rADC increased in the three areas in the early stage of reperfusion (1 hour after the reperfusion). However, the rate of rADC improvement was significantly slower in IG than in IC and RP. Different temporal evolutions of rFA were observed in the three areas in the following stage of reperfusion (3-24 hours after the reperfusion), which continued to decline in IG but slightly elevated in IC and RP. DISCUSSION: These findings suggest that the evolution of DTI parameters can help in the assessment of cerebral ischemia/reperfusion injury in the penumbra and predict the growth of the infarction area after stroke.

[Toxicity of Caowu on in vitro cultured rat embryos].

OBJECTIVE: To study the developmental toxicity of Caowu and its mechanism on rat embryos. METHODS: Whole embryo culture was applied to test the effects of Caowu on rat embryos during the critical period of organogenesis. All embryos isolated on gestational day 9.5 were exposed to different concentrations of Caowu. The growth and differentiation of the embryos at the end of the 48 h culture period were observed. RESULTS: The embryonic growth and development were adversely affected by Caowu at the concentration of 2.5 mg/mL, which were represented as reduced yolk sac diameters, decreased number of somites, and lower morphological scores. With the increase of the concentrations of Caowu, more severe teratogenic effects appeared, including cardiac defect (undivided cardiac tube and inflated pericardial cavity), irregular somites, and brain malformation such as narrow brain vesicles. CONCLUSION: Caowu has direct embryotoxic effects on rats during the organogenesis period. We suggest that pregnant women in the first trimester use Caowu with cautions before a conclusion has been made.

[Effects of ErbB signal and protein kinase B on monkey cardiocyte apoptosis induced by rapid pacing].

This study is to investigate the roles of neuregulin receptor ErbB2 and protein kinase B (PKB) in pacing-induced heart failure of rhesus monkey. Rapid pacing was used to induce heart failure in rhesus monkey. Aorta intubatton was used to perform hemodynamic measurements, 17 days after pacing. N-Terminal pro-brain natriuretic peptide (BNP), one of the most important molecular marker of heart failure, was also measured by the method of electrochemical luminescence immunoassay. The mRNA expressions of ErbB2, PKB and Bcl-xl were detected in the left ventricular free walls by RT-PCR method. The expressions of phospho-PKB and Bcl-xl on protein level were also detected by Western blotting. The contractibility of cardiac muscle decreased significantly, which consisted with the increase of BNP. Compared with control group, the mRNA expressions of ErbB2, PKB and Bcl-xl were depressed, and similar results were also found in the protein expression analysis of phospho-PKB and Bcl-xl. The expressions of ErbB2, PKB and Bcl-xl were down-regulated during heart failure in rhesus monkey which suggested the important roles of ErbB2 receptor and PKB in the mechanism of heart failure.

[Effects of recombined human neuregulin on the contractibility of cardiac muscles of rhesus monkeys with pacing-induced heart failure].

OBJECTIVE: To evaluate the effects of recombined human Neuregulin on the contractibility of cardiac muscles of Rhesus Monkeys with pacing-induced heart failure and to reveal the possible mechanisms involved. METHODS: Twenty four rhesus monkeys were randomly divided into three groups (shame operated group, heart failure group and Neuregulin treated group), each with 8 monkeys. Heart failures were induced by rapid pacing (240 heartbeats/min). Daily intravenous injection of recombined human Neuregulin [3 microg/(kg x d)] and medical salt fluid were given to the monkeys for 10 days for the Neuregulin treated group and heart failure group respectively. Hemodynamic measurements such as peak positive rate of change in left ventricular blood pressure (+dP/dtmax) and left ventricular systolic, and end-diastolic blood pressures (LVSP and LVEDP) were compared between groups. The real-time quantitative RT-PCR was undertaken to detect the expression of myosin heavy chain mRNA in the left ventricular cardiac muscle. RESULTS: The monkeys in the heart failure group had lower levels of +dP/dtmax and LVSP and higher levels of LVEDP than those in the shame operated group (P < 0.05). The monkeys in the Neuregulin treated group had higher levels of + dP/dtmax than those in the heart failure group (P < 0.05). Lower expression of alpha-myosin heavy chain mRNA in the heart failure group was found compared with the shame operated group and Neuregulin treated group (P < 0.05). CONCLUSION: Recombined human Neuregulin can enhance the contractibility of cardiac muscles and relieve heart failure syndrome through reversing the falling of alpha-myosin heavy chain induced by rapid ventricular pacing.