pH measurement as quality control on human post mortem brain tissue: a study of the BrainNet Europe consortium

Aims:  Most brain diseases are complex entities. Although animal models or cell culture experiments mimic some disease aspects, human post mortem brain tissue remains essential to advance our understanding of brain diseases using biochemical and molecular techniques. Post mortem artefacts must be properly understood, standardized, and either eliminated or factored into such experiments. Here we examine the influence of several premortem and post mortem factors on pH, and discuss the role of pH as a biochemical marker for brain tissue quality. Methods:  We assessed brain tissue pH in 339 samples from 116 brains provided by 8 different European and 2 Australian brain bank centres. We correlated brain pH with tissue source, post mortem delay, age, gender, freezing method, storage duration, agonal state and brain ischaemia. Results:  Our results revealed that only prolonged agonal state and ischaemic brain damage influenced brain tissue pH next to repeated freeze/thaw cycles. Conclusions:  pH measurement in brain tissue is a good indicator of premortem events in brain tissue and it signals limitations for post mortem investigations.     

Heavy alcohol consumption and neuropathological lesions: A post‐mortem human study

Epidemiological studies have indicated that excessive alcohol consumption leads to cognitive impairment, but the specific pathological mechanism involved remains unknown. The present study evaluated the association between heavy alcohol intake and the neuropathological hallmark lesions of the three most common neurodegenerative disorders, i.e., Alzheimer's disease (AD), dementia with Lewy bodies (DLB), and vascular cognitive impairment (VCI), in post‐mortem human brains. The study cohort was sampled from the subjects who underwent a medicolegal autopsy during a 6‐month period in 1999 and it included 54 heavy alcohol consumers and 54 age‐ and gender‐matched control subjects. Immunohistochemical methodology was used to visualize the aggregation of β‐amyloid, hyperphosphorylated τ, and α‐synuclein and the extent of infarcts. In the present study, no statistically significant influence was observed for alcohol consumption on the extent of neuropathological lesions encountered in the three most common degenerative disorders. Our results indicate that alcohol‐related dementia differs from VCI, AD, and DLB; i.e., it has a different etiology and pathogenesis. © 2009 Wiley‐Liss, Inc.     

RNA integrity in post mortem human variant Creutzfeldt-Jakob disease (vCJD) and control brain tissue

K. R. Sherwood, M. W. Head, R. Walker, C. Smith, J. W. Ironside and J. K. Fazakerley (2011) Neuropathology and Applied Neurobiology 37, 633–642 RNA integrity in post mortem human variant Creutzfeldt–Jakob disease (vCJD) and control brain tissue Aims: To determine premortem and post mortem factors affecting quality and yield of RNA isolated from the unique archived brain material in the UK National Creutzfeldt–Jakob Disease Surveillance Unit Brain and Tissue Bank and to compare this to control brain tissue with no neurological disease. Methods: In parallel and in replicate, RNA was prepared from the frontal parasagittal or subfrontal cortex of samples dissected from half brains (frozen intact) or from brain samples snap frozen or placed in RNALater. A total of 350 RNA samples from 78 human autopsy cases, 21 variant Creutzfeldt–Jakob disease, 26 other neurological diseases and 31 non‐neurological diseases were studied. Results: There was no difference in the quality or yield of RNA isolated from variant Creutzfeldt–Jakob disease, other neurological disease and non‐neurological disease brains. RNA preparations from archived frozen half brains or snap frozen autopsy samples were generally of poor quality (RNA integrity number < 5). There was a highly significant negative correlation between the number of times frozen half brains had been sampled and the quality of RNA. Samples stored in RNALater provided higher‐quality RNA (RNA integrity number > 5). Age at death, gender, post mortem interval and freezer storage time had no effect on RNA quality. Conclusion: Reasonable‐quality RNA can be isolated from samples dissected from archived frozen human half brains but repeated sampling results in RNA degradation. Better‐quality RNA is obtained from samples placed in RNALater than from snap frozen samples. The quality and yield of RNA are not affected by age at death, gender, post mortem interval of >6 h or freezer storage time.     

Brain‐derived neurotrophic factor as a biomarker for mood disorders: An historical overview and future directions

Mood disorders, such as major depressive disorder (MDD) and bipolar disorder (BPD), are the most prevalent psychiatric conditions, and are also among the most severe and debilitating. However, the precise neurobiology underlying these disorders is currently unknown. One way to combat these disorders is to discover novel biomarkers for them. The development of such biomarkers will aid both in the diagnosis of mood disorders and in the development of effective psychiatric medications to treat them. A number of preclinical studies have suggested that the brain‐derived neurotrophic factor (BDNF) plays an important role in the pathophysiology of MDD. In 2003, we reported that serum levels of BDNF in antidepressant‐naive patients with MDD were significantly lower than those of patients medicated with antidepressants and normal controls, and that serum BDNF levels were negatively correlated with the severity of depression. Additionally, we found that decreased serum levels of BDNF in antidepressant‐naive patients recovered to normal levels associated with the recovery of depression after treatment with antidepressant medication. This review article will provide an historical overview of the role played by BDNF in the pathophysiology of mood disorders and in the mechanism of action of therapeutic agents. Particular focus will be given to the potential use of BDNF as a biomarker for mood disorders. BDNF is initially synthesized as a precursor protein proBDNF, and then proBDNF is proteolytically cleaved to the mature BDNF. Finally, future perspectives on the use of proBDNF as a novel biomarker for mood disorders will be discussed.     

Determination of monoamine and monoamine metabolites in the human brain: post mortem studies in a group of suicides and in a control group.

Different nuclei and regions of the brain from patients who had committed suicide and from controls were analysed for their content of monoamine and monoamine metabolites. There was a post mortem breakdown of 5-hydroxyindoleacetic acid (5-HIAA) which could be correlated to the time elapsed between the occurrence of death and autopsy. Homovanillic acid (HVA) and the monoamines did not decrease post mortem during the time observed (6-148 hours). There was no significant correlation between age and chemical variables in this investigation. There were no significant group differences between suicides and controls concerning dopamine, noradrenaline, 5-hydroxytryptamine, and HVA, 5-HIAA levels were significantly lower in the suicide group in six out of eight parts of the brain investigated. It was, however, also demonstrated that there was a longer time elapse between the occurrence of death and autopsy in the suicide group. The suicides came on average 48 hours later to autopsy than the controls. As there was a post mortem decrease of 5-HIAA, this time variable had to be kept constant when group differences were analysed. When the influence of this time variable was eliminated there were no longer any differences between suicides and controls. According to this investigation, there seem to be no differences in levels of monoamines and their metabolites between suicides and controls.     

Functional organization of the human posterior cingulate cortex,revealed by multiple connectivity‐based parcellation methods

Based on cytoarchitecture, the posterior cingulate cortex (PCC) is thought to be comprised of two distinct functional subregions: the dorsal and ventral PCC (dPCC and vPCC). However, functional subregions do not completely match anatomical boundaries in the human brain. To understand the relationship between the functional organization of regions and anatomical features, it is necessary to apply parcellation algorithms based on functional properties. We therefore defined functionally informed subregions in the human PCC by parcellation of regions with similar patterns of functional connectivity in the resting brain. We used various patterns of functional connectivity, namely local, whole‐brain and diffuse functional connections of the PCC, and various clustering methods, namely hierarchical, spectral, and k‐means clustering to investigate the subregions of the PCC. Overall, the approximate anatomical boundaries and predicted functional regions were highly overlapped to each other. Using hierarchical clustering, the PCC could be clearly separated into two anatomical subregions, namely the dPCC and vPCC, and further divided into four subregions segregated by local functional connectivity patterns. We show that the PCC could be separated into two (dPCC and vPCC) or four subregions based on local functional connections and hierarchical clustering, and that subregions of PCC display differential global functional connectivity, particularly along the dorsal‐ventral axis. These results suggest that differences in functional connectivity between dPCC and vPCC may be due to differences in local connectivity between these functionally hierarchical subregions of the PCC. Hum Brain Mapp 38:2808–2818, 2017. © 2017 Wiley Periodicals, Inc.     

DPP4‐inhibitor improves neuronal insulin receptor function,brain mitochondrial function and cognitive function in rats with insulin resistance induced by high‐fat diet consumption

High‐fat diet (HFD) consumption has been demonstrated to cause peripheral and neuronal insulin resistance, and brain mitochondrial dysfunction in rats. Although the dipeptidyl peptidase‐4 inhibitor, vildagliptin, is known to improve peripheral insulin sensitivity, its effects on neuronal insulin resistance and brain mitochondrial dysfunction caused by a HFD are unknown. We tested the hypothesis that vildagliptin prevents neuronal insulin resistance, brain mitochondrial dysfunction, learning and memory deficit caused by HFD. Male rats were divided into two groups to receive either a HFD or normal diet (ND) for 12 weeks, after which rats in each group were fed with either vildagliptin (3 mg/kg/day) or vehicle for 21 days. The cognitive function was tested by the Morris Water Maze prior to brain removal for studying neuronal insulin receptor (IR) and brain mitochondrial function. In HFD rats, neuronal insulin resistance and brain mitochondrial dysfunction were demonstrated, with impaired learning and memory. Vildagliptin prevented neuronal insulin resistance by restoring insulin‐induced long‐term depression and neuronal IR phosphorylation, IRS‐1 phosphorylation and Akt/PKB‐ser phosphorylation. It also improved brain mitochondrial dysfunction and cognitive function. Vildagliptin effectively restored neuronal IR function, increased glucagon‐like‐peptide 1 levels and prevented brain mitochondrial dysfunction, thus attenuating the impaired cognitive function caused by HFD.     

Identification of extrastriatal dopamine D2 receptors in post mortem human brain with [I]epidepride

The regional distribution of striatal and extrastriatal dopamine D₂ receptors in human brain was studied in vitro with(S)-N-[(1-ethyl-2-pyrrolidinyl)methyl]-5-[¹²⁵I]iodo-2,3-dimethoxybenzamide, [¹²⁵I]epidepride, using post mortem brain specimens from six subjects. Scatchard analysis of the saturation equilibrium binding in twenty-three regions of post mortem brain revealed highest levels of binding in the caudate (16.5 pmol/g tissue) and putamen (16.6 pmol/g tissue) with lower levels seen in the globus pallidus (7.0 pmol/g tissue), nucleus accumbens (7.2 pmol/g tissue), hypothalamus (1.8 pmol/g tissue), pituitary (1.3 pmol/g tissue), substantia innominata (1.0 pmol/g tissue), and amygdala (0.87 pmol/g tissue). Of note was the presence of dopamine D₂ receptors in the four thalamic nuclei studied, i.e. anterior nucleus (1.0 pmol/g tissue), dorsomedial nucleus (0.96 pmol/g tissue), ventral nuclei (0.72 pmol/g tissue), and pulvinar (0.86 pmol/g tissue), at levels comparable to the amygdala (0.87 pmol/g tissue) and considerably higher than levels seen in anterior cingulate (0.26 pmol/g tissue) or anterior hippocampus (0.36 pmol/g tissue). The frontal cortex had very low levels of dopamine D₂ receptors (0.17–0.20 pmol/g tissue) while the inferior and medial temporal cortex had relatively higher levels (0.31–0.46 pmol/g tissue). Inhibition of [¹²⁵I]epidepride binding by a variety of neurotransmitter ligands to striatal, ventral thalamic and inferior temporal cortical homogenates demonstrated that [¹²⁵I]epidepride binding was potently inhibited only by dopamine D₂ ligands. The present study demonstrates that dopamine D₂ receptors are present in basal ganglia, many limbic regions, cortex and in the thalamus. The density of thalamic D₂ receptors is comparable to many limbic regions and is considerably higher than in cortex. Very few frontal lobe D₂ receptors are present in man.     

Impact of correction factors in human brain lesion‐behavior inference

Statistical voxel‐based lesion‐behavior mapping (VLBM) in neurological patients with brain lesions is frequently used to examine the relationship between structure and function of the healthy human brain. Only recently, two simulation studies noted reduced anatomical validity of this method, observing the results of VLBM to be systematically misplaced by about 16 mm. However, both simulation studies differed from VLBM analyses of real data in that they lacked the proper use of two correction factors: lesion size and “sufficient lesion affection.” In simulation experiments on a sample of 274 real stroke patients, we found that the use of these two correction factors reduced misplacement markedly compared to uncorrected VLBM. Apparently, the misplacement is due to physiological effects of brain lesion anatomy. Voxel‐wise topographies of collateral damage in the real data were generated and used to compute a metric for the inter‐voxel relation of brain damage. “Anatomical bias” vectors that were solely calculated from these inter‐voxel relations in the patients' real anatomical data, successfully predicted the VLBM misplacement. The latter has the potential to help in the development of new VLBM methods that provide even higher anatomical validity than currently available by the proper use of correction factors. Hum Brain Mapp 38:1692–1701, 2017. © 2017 Wiley Periodicals, Inc.     

Hypoxic‐ischemic brain injury exacerbates neuronal apoptosis and precipitates spontaneous seizures in glucose transporter isoform 3 heterozygous null mice

We examined the effects of 45‐min hypoxia (FiO₂ 0.08; Hx) vs. normoxia (FiO₂ 0.21; Nx) on the ipsilateral (Ipsi) and contralateral (Ctrl) sides of the brain in neuronal glucose transporter isoform 3 (Glut3) heterozygous null mice (glut3^+/−) and their wild‐type littermates (WT), undergoing unilateral carotid artery ligation. Glut3^+/− mice, under Nx, demonstrated a compensatory increase in blood–brain barrier/glial Glut1 protein concentration and a concomitant increase in neuronal nitric oxide synthase (nNOS) enzyme activity and Bax protein, with a decrease in procaspase 3 protein (P < 0.05 each). After Hx, reoxygenation in FiO₂ of 0.21 led to no comparable adaptive up‐regulation of the ipsilateral brain Glut3 or Glut1 protein at 4 hr and Glut1 at 24 hr in glut3^+/− vs. WT. These brain Glut changes in glut3^+/− but not WT mice were associated with an increase in proapoptotic Bax protein and caspase‐3 enzyme activity (P < 0.01 each) and a decline in the antiapoptotic Bcl‐2 and procaspase‐3 proteins (P < 0.05 each). Glut3^+/− mice after Hx demonstrated TUNEL‐positive neurons with nuclear pyknosis in most ipsilateral (hypoxic‐ischemia) brain regions. A subset (∼55%) of glut3^+/− mice developed spontaneous seizures after hypoxic‐ischemia, confirmed by electroencephalography, but the WT mice remained seizure‐free. Pentylenetetrazole testing demonstrated an increased occurrence of longer lasting clinical seizures at a lower threshold in glut3^+/− vs. WT mice, with no detectable differences in monamine neurotransmitters. We conclude that hypoxic‐ischemic brain injury in glut3^+/− mice exacerbates cellular apoptosis and necrosis and precipitates spontaneous seizures. © 2010 Wiley‐Liss, Inc.