In situ hybridization for vasopressin mRNA in the human supraoptic and paraventricular nucleus; quantitative aspects of formalin-fixed paraffin-embedded tissue sections as compared to cryostat sections.

In order to study the suitability of formalin-fixed paraffin-embedded brain tissue for vasopressin (AVP)-mRNA detection, we used symmetric halves of 5 human hypothalami. In every case, one half was formalin fixed for 10-35 days and paraffin embedded while the other half was frozen rapidly. Following in situ hybridization (ISH) histochemistry on systematically obtained sections of the supraoptic (SON) and paraventricular nucleus (PVN) of both halves, total amounts of AVP-mRNA in these nuclei were estimated using densitometry of film autoradiographs. Total amounts of radioactivity were found to vary considerably between patients and amounted to 1297 +/- 302 arbitrary units (AU) (PVN) (mean +/- SEM) and 2539 +/- 346 (SON) for the cryostat sections and 868 +/- 94 (PVN) and 1259 +/- 126 (SON) for the paraffin tissue. Variations introduced by the method itself yielded a coefficient of variation of only 0.19. Furthermore, a non-significant negative trend with postmortem delay was found in cryostat tissue, but not in paraffin sections. No effect of fixation time was observed in the paraffin tissue. Both ways of tissue treatment have specific advantages and disadvantages that may be different for other probes or other brain areas. For ISH of a highly abundant mRNA like AVP in a very heterogeneous brain area such as the human hypothalamus, formalin-fixed paraffin-embedded tissue sections can be used for quantitative analysis of entire brain nuclei because of the small variation in this tissue, the remarkably good signal recovery (some 75% as compared to cryostat sections) and its practical advantages with regards to anatomical orientation, storage and sampling of the tissue.

Corticotropin-releasing hormone mRNA levels in the paraventricular nucleus of patients with Alzheimer's disease and depression.

OBJECTIVE: Greater activity of the hypothalamic-pituitary-adrenal (HPA) axis is associated with specific neurological and psychiatric disorders, including Alzheimer's disease and depression. Hyperactivation of paraventricular corticotropin-releasing hormone (CRH) neurons may form the basis of this increased activity of the HPA axis. METHOD: Activation of the CRH neurons was determined through measurement of the amount of CRH-mRNA in the paraventricular nucleus by using quantitative, in situ hybridization histochemistry with systematically sampled frontal sections through the hypothalamus of routinely formalin-fixed and paraffin-embedded autopsy brain material of 10 comparison subjects, 10 patients with Alzheimer's disease, and seven depressed patients. RESULTS: CRH-mRNA levels in the paraventricular nucleus of Alzheimer's patients were markedly higher than those of comparison subjects, whereas CRH-mRNA levels in the paraventricular nucleus of depressed patients were even higher than the levels of Alzheimer's patients. CONCLUSIONS: Paraventricular CRH neurons in Alzheimer's disease and depression are hyperactivated, and this hyperactivation may contribute to the etiology of these disorders.

Increased number of corticotropin-releasing hormone expressing neurons in the hypothalamic paraventricular nucleus of patients with multiple sclerosis.

Observations in experimental allergic encephalomyelitis (EAE), a model for multiple sclerosis (MS), have indicated that a low activity of the hypothalamo-pituitary-adrenal (HPA) system is accompanied by a high susceptibility for EAE in rat strains and that elevated corticosteroid levels are necessary for spontaneous recovery from EAE. The HPA axis activity is regulated by both corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP). Both types of neurons are localized in the paraventricular nucleus (PVN) of the hypothalamus. We determined the number of immunocytochemically identified CRH-immunoreactive (CRH-IR) and AVP-immunoreactive (AVP-IR) neurons in the PVN of the human hypothalamus of 8 MS patients, aged 34-63 years, and 8 age-matched control subjects without any primary neurological or psychiatric disorders, aged 30-59 years. In addition, the number of oxytocin (OXT) immunoreactive (OXT-IR) neurons was determined, since these neurons innervate brain stem nuclei and might thus be related to autonomic disturbances in MS. In MS the staining intensity for AVP was clearly lower and for OXT slightly lower. For CRH, the staining intensity was similar in both groups, and, moreover, in MS patients the number of CRH-IR cells in the PVN was found to be about 2.4 times higher than that in the control group. The number of OXT-IR or AVP-IR cells in the PVN of MS patients was not significantly different from that of the control group. Our results point to an activation of the neuroendocrine HPA axis which may be compatible with the idea that the HPA axis is involved in recovery from MS.(ABSTRACT TRUNCATED AT 250 WORDS)

In situ hybridization for vasopressin mRNA in the human supraoptic and paraventricular nucleus; quantitative aspects of formalin-fixed paraffin-embedded tissue sections as compared to cryostat sections.

In order to study the suitability of formalin-fixed paraffin-embedded brain tissue for vasopressin (AVP)-mRNA detection, we used symmetric halves of 5 human hypothalami. In every case, one half was formalin fixed for 10-35 days and paraffin embedded while the other half was frozen rapidly. Following in situ hybridization (ISH) histochemistry on systematically obtained sections of the supraoptic (SON) and paraventricular nucleus (PVN) of both halves, total amounts of AVP-mRNA in these nuclei were estimated using densitometry of film autoradiographs. Total amounts of radioactivity were found to vary considerably between patients and amounted to 1297 +/- 302 arbitrary units (AU) (PVN) (mean +/- SEM) and 2539 +/- 346 (SON) for the cryostat sections and 868 +/- 94 (PVN) and 1259 +/- 126 (SON) for the paraffin tissue. Variations introduced by the method itself yielded a coefficient of variation of only 0.19. Furthermore, a non-significant negative trend with postmortem delay was found in cryostat tissue, but not in paraffin sections. No effect of fixation time was observed in the paraffin tissue. Both ways of tissue treatment have specific advantages and disadvantages that may be different for other probes or other brain areas. For ISH of a highly abundant mRNA like AVP in a very heterogeneous brain area such as the human hypothalamus, formalin-fixed paraffin-embedded tissue sections can be used for quantitative analysis of entire brain nuclei because of the small variation in this tissue, the remarkably good signal recovery (some 75% as compared to cryostat sections) and its practical advantages with regards to anatomical orientation, storage and sampling of the tissue.

Increased cortisol levels in aging and Alzheimer's disease in postmortem cerebrospinal fluid.

The hypothalamo-pituitary-adrenal (HPA) axis is activated during aging and even more so in dementia. Increased levels of corticosteroids may be neurotoxic. Therefore we have investigated cortisol levels in cerebrospinal fluid (CSF) of Alzheimer patients and controls. Ventricular postmortem CSF was collected from clinically and neuropathologically well-defined Alzheimer patients (n = 26) and control subjects (n = 21). In the group of Alzheimer patients the mean CSF total cortisol level was 83% higher than that in the controls. In presenile Alzheimer patients (< 65 years of age; n = 13) the CSF-cortisol level was 5 times higher than that of presenile controls (n = 7). In contrast, senile Alzheimer patients (n = 13) and controls of over 65 years of age (n = 14) did not show a significant difference in CSF-cortisol levels. The presence or absence of a difference in the cortisol-CSF levels in, respectively, presenile or senile Alzheimer patients as compared to controls was due to the 3.5-fold rise of CSF-cortisol in control subjects over 65 years of age as compared with controls under 65 years of age. The CSF-cortisol levels in presenile and senile Alzheimer patients were similar. No significant correlation was observed in the Alzheimer patients between age of onset of the dementia and CSF cortisol levels or duration of Alzheimer's disease and CSF cortisol levels. The finding that in senile Alzheimer patients cortisol levels were similar to those of unaffected age-matched controls does not seem to support the cortisol neurotoxicity hypothesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Colocalization of tyrosine hydroxylase with oxytocin or vasopressin in neurons of the human paraventricular and supraoptic nucleus.

In the developing and adult human paraventricular (PVN) and supraoptic (SON) nucleus, a large proportion of neurons contains the catecholamine-synthesizing enzyme tyrosine hydroxylase (TH). In the present study we investigated the possible colocalization of TH with oxytocin (OXT) or vasopressin (VP) in the adult and neonatal PVN and SON. Adjacent paraffin sections were incubated simultaneously with two antibodies: a polyclonal against TH and a monoclonal against OXT or VP and stained with a double peroxidase-antiperoxidase/alkaline phosphatase method. We observed that TH-immunoreactive(IR) perikarya in the human PVN and SON were also positive for OXT or VP. A clear difference between the neonates and adult cases of our sample was observed in the proportion of TH-IR neurons that colocalize OXT or VP. In the neonates the majority of the TH-IR perikarya was also stained for VP, while only few TH-IR neurons were also positive for OXT. The opposite was observed in the adults, where the majority of the double-stained TH-IR neurons colocalizes OXT while only few TH-IR perikarya appear to contain VP. Our study establishes the colocalization of TH with OXT or VP in the adult and neonatal PVN and SON and indicates that antemortem factors such as perinatal hypoxia might increase TH-immunoreactivity of the VP neurons in man.

Increased numbers of corticotropin-releasing hormone expressing neurons in the hypothalamic paraventricular nucleus of depressed patients.

The hypothalamo-pituitary-adrenal (HPA) axis is known to be activated in depressed patients. Although direct evidence is lacking, this activation is hypothesized to be due to hyperactivity of corticotropin-releasing hormone (CRH) neurons of the hypothalamic paraventricular nucleus (PVN). Recent immunocytochemical studies in experimental animals and in humans showed that the number of CRH-expressing neurons correlated with the activity of these neurons. In addition, colocalization of AVP in CRH neurons has been shown to be an index for the secretory activity. Therefore, we estimated the total number of CRH-immunoreactive neurons and their fraction showing colocalization with AVP in the PVN of 10 control subjects and of 6 depressed patients who were diagnosed to be suffering from a major depression or a bipolar disorder. The mean total number of CRH-expressing neurons of the 6 depressed patients was four times higher, and the number of CRH neurons co-expressing AVP was almost three times higher than those in the control group. We also determined the two activity parameters of CRH neurons in the PVN of 2 subjects with a depressive organic mood syndrome or a depressive disorder not otherwise specified. In these two 'non-major depressed' subjects, the activity parameters of CRH neurons were comparable to those of control subjects. Our observations strongly support the hypothesis that CRH neurons in the PVN are hyperactivated in major depressed patients. This hyperactivity might be causally related to at least part of the symptomatology of depression.

Similar age related increase of vasopressin colocalization in paraventricular corticotropin-releasing hormone neurons in controls and Alzheimer patients.

Recent studies on experimental animals showed that long term activation of the hypothalamo-pituitary-adrenal axis is associated with increased vasopressin (AVP) colocalization in paraventricular corticotropin-releasing hormone (CRH) neurons. In the present study we estimated the fraction of CRH neurons in which AVP is colocalized by double label immunocytochemistry in hypothalami of 10 control subjects of 21-91 years of age and 10 age-matched Alzheimer patients. CRH neurons in the paraventricular nucleus (PVN) of Alzheimer patients and control subjects showed similar age dependent increases in AVP colocalization. Based on this parameter, it seems that CRH neurons of Alzheimer patients are not overactivated as compared to age-matched controls, but e.g. changes in m-RNA for CRH should still be established.

Age-related increase in the total number of corticotropin-releasing hormone neurons in the human paraventricular nucleus in controls and Alzheimer's disease: comparison of the disector with an unfolding method.

It has been hypothesized that the corticotropin-releasing hormone (CRH) neurons of the hypothalamic paraventricular nucleus (PVN) become hyperactive with age, and even more so in Alzheimer's disease. This hyperactivity could be due to an increased production of CRH per neuron, or an increased number of PVN neurons producing CRH, or both. As a first step in elucidating which of these biological mechanisms might be operative, we have estimated the absolute number of CRH immunoreactive neurons in the PVN of 10 human control subjects between 36 and 91 years of age and 10 Alzheimer patients between 40 and 97 years of age. CRH neurons were immunocytochemically detected in 6 microns paraffin sections with the aid of a highly specific monoclonal antibody to CRH. The antibody signal was amplified by the biotin-streptavidin and alkaline phosphatase methods. The absolute number of CRH neurons in the PVN was obtained by multiplying the number of CRH neurons in a unit volume (NV) by the total volume of the PVN. Two different methods were used to estimate the NV: an unfolding method and a disector method (about three times more time-consuming). Compared to the disector, the unfolding method consistently yielded a lower cell number for all patients by 38% (+/- 2.8%; mean +/- SEM). However, both methods yielded an increase in the absolute number of CRH neurons in control and Alzheimer patients with age. No statistically significant difference in the absolute number of CRH neurons was found between control and Alzheimer patients with both methods. The age-dependent increase in the absolute number of CRH neurons within the PVN of both control and Alzheimer patients is interpreted as a sign of activation of the CRH neurons with age.

Localization of corticotropin-releasing hormone (CRH) neurons in the paraventricular nucleus of the human hypothalamus; age-dependent colocalization with vasopressin.

Immunocytochemical staining, using a monoclonal antibody against corticotropin-releasing hormone, was performed on hypothalami of 13 human subjects between 23 and 91 years of age who had not suffered from a primary neurological or psychiatric disease. Corticotropin-releasing hormone (CRH) immunoreactivity was present in neurons of the paraventricular nucleus (PVN) and in their fibers running to the median eminence. The CRH-positive neurons were scattered throughout the PVN, but in the rostral part relatively few cells were present. There were large individual differences in the number and staining intensity of CRH neurons in the PVN and in the staining intensity of the median eminence. These differences seemed not to be attributed to age, sex, postmortem delay, fixation time or hour of death. In the rat, too, no relationship was found between a postmortem delay of up to 24 h and CRH staining intensity of the median eminence. Since the distribution of CRH-immunoreactive neurons in the human PVN strongly overlap with vasopressin, colocalization of these peptides was investigated in a double label study and indeed found in subjects ranging between 43 and 91 years of age. However, cells staining for only one of the peptides were also observed. The vasopressin cells had a mean cellular profile area which was 2.3 times as large as the CRH cells and 2.2 times as large as the CRH and vasopressin containing neurons. In younger subjects (23-37 years of age) no colocalization of the two peptides was seen. The age-dependent colocalization of CRH with vasopressin is interpreted as a sign of increased activation of the CRH neurons with age.

Functional neuroanatomy and neuropathology of the human hypothalamus.

The human hypothalamus is involved in a wide range of functions in the developing, adult and aging subject and is responsible for a large number of symptoms of neuroendocrine, neurological and psychiatric diseases. In the present review some prominent hypothalamic nuclei are discussed in relation to normal development, sexual differentiation, aging and a number of neuropathological conditions. The suprachiasmatic nucleus, the clock of the brain, shows seasonal and circadian variations in its vasopressin neurons. During normal aging, but even more so in Alzheimer's disease, the number of these neurons decreases. In homosexual men this nucleus is larger than in heterosexual men. The difference between the sexually dimorphic nuclei of men and women arises between the ages of 2-4 to puberty. In adult men this nucleus is twice as large as in adult women. In the process of aging, a sex-dependent decrease in cell number occurs. The vasopressin and oxytocin cells of the supraoptic and paraventricular nucleus are present in adult numbers as early as mid-gestation. Lower oxytocin neuron numbers are found in Prader-Willi syndrome, AIDS and Parkinson's disease. Familial hypothalamic diabetes insipidus is based upon a point mutation in the vasopressin-neurophysin-glycopeptide gene. Parvicellular corticotropin-releasing hormone-containing neurons in the paraventricular nucleus increase in number and are activated during the course of aging. In post-menopausal women, the infundibular or arcuate nucleus contains hypertrophic neurons containing oestrogen receptors. These neurons may be involved in the initiation of menopausal flushes. The nucleus tuberalis lateralis may be involved in feeding behaviour and metabolism. In Huntington's disease the majority of its neurons is lost; in Alzheimer's disease it shows very strong cytoskeletal alterations. Tuberomammillary nucleus neurons contain, e.g., histamine or galanine, and project to the cortex. Strong cytoskeletal changes, as well as plaques and tangles are found in this nucleus in Alzheimer's disease. The various hypothalamic nuclei are probably involved in many functions and symptoms of which only a minority has been revealed.

Detection of single-strand breaks and base damage in DNA of blood cells from leukaemia patients receiving chemo- and radiotherapy.

Chemotherapy combined with total-body irradiation (TBI), a conditioning regimen for bone-marrow transplantation (BMT), causes lesions in the cellular DNA of the patients treated. To understand possible consequences of the DNA damage induced during such treatment, information is required about the nature of the damage, the level of induction and its persistence, and about the importance of the various lesions for cell-lethality and/or mutation induction. Recently, we developed a sensitive immunochemical method to quantify single-strand breaks (SSB) in the DNA of mammalian cells. In addition, a modification of the so-called alkaline elution technique was introduced which allows quantification of SSB together with base damage (SSB+BD). These methods have now been applied successfully to study the in vivo induction and repair of DNA damage in WBC of leukaemia patients who prior to BMT were treated with cyclophosphamide (CY) and received TBI. SSB and SSB+BD were determined after two treatments with CY (60 mg kg-1) followed by TBI (4.5-8.6Gy). The CY treatments gave rise to rather persistent SSB. In addition to these, radiation-induced SSB and SSB+BD could be detected shortly after TBI. However, 105 min after TBI, these SSB could be observed no longer, as a result of rapid repair.

Development of Neurons and Glial Cells in Cerebral Cortex, Cultured in the Presence or Absence of Bioelectric Activity: Morphological Observations.

Chronic blockade of bioelectric activity (BEA) has been shown to increase neuronal cell death in tissue culture, but the effects of this treatment on non-neuronal cells have not been investigated. To determine which cell types are affected by chronic suppression of BEA, we investigated their morphological development in primary cultures of rat cerebral cortex, grown with or without the sodium channel blocker tetrodotoxin (TTX). Morphological development was monitored by phase-contrast microscopy and by immunofluorescent staining of markers specific for neurons (NSE, MAP2, B-50, and the 200 kD neurofilament protein), astrocytes (GFAP), oligodendrocytes (galactocerebroside), macrophages (ED-1) and fibroblasts (fibronectin). Neurons in control cultures steadily increased in size and elaborated a dense network of axons and dendrites during the first 3 weeks. Astrocytes proliferated strongly and formed a 'bottom-layer' on which other cells grew. Part of the astrocytes migrated into the peripheral area of the culture, but retracted to the centre after 14 days in vitro (DIV). Oligodendrocytes and macrophages also increased in number, but oligodendrocytes were completely lost by 28 DIV. After 3 weeks, axons that had grown into the periphery of the culture gradually retracted and/or degenerated, following the retracting astrocytes. Some of the neurons died after 21 DIV, but a large part persisted until 42 DIV. Upon TTX treatment from 5/6 DIV, cultures with few macrophages showed an increase in the proportion of necrotic nuclei at 14 and 21 DIV. The retraction of peripherally located fibres was accelerated by 3 - 4 days and their degeneration was augmented. Neuronal density decreased to zero between 21 and 42 DIV. Astrocytes showed a clear decrease in density from 28 DIV. Conversely, the density of macrophages was increased about two-fold from 14 DIV. These results indicate that both neurons and glia are affected by chronic TTX treatment.