Quantitative assessment of relative changes of immunohistochemical staining by light microscopy in specified anatomical regions.

Despite the advent of ever newer microscopic techniques for the study of the distribution of macromolecules in biological tissues, the enzyme-based immunohistochemical (IHC) methods are still used widely and routinely. However, the acquisition of reliable conclusions from the pattern of the reaction products of IHC procedures is hindered by the regular need for subjective judgments, in view of frequent inconsistencies in staining intensity from section to section or in repeated experiments. Consequently, when numerical comparisons are required, light microscopic morphological descriptions are commonly supplemented with analytical data (e.g. from Western blot analyses); however, these cannot be directly associated with accurate structural information and can easily be contaminated with data from outside the region of interest. Alternatively, to eliminate the more or less subjective evaluation of the results of IHC staining, procedures should be developed that correct for the variability of staining through the use of objective criteria. This paper describes a simple procedure, based on digital image analysis methods and the use of an internal reference area on the analyzed sections, that reduces the operator input and hence subjectivity, and makes the relative changes in IHC staining intensity in different experiments comparable. The reference area is situated at a position of the section that is not affected by the experimental treatment, or a disease condition, and that can therefore be used to specify the baseline of the IHC staining. Another source of staining variability is the internal heterogeneity of the object to be characterized, which means that identical fields can never be analyzed. To compensate for this variability, details are given of a systematic random sampling paradigm, which provides simple numerical data describing the extent and strength of IHC staining throughout the entire volume to be characterized. In this integrated approach, the figures are derived by pooling relative IHC staining intensities from all sections of the series from a particular animal. The procedure (1) eliminates the problem arising from the personal assessment of the significance of the IHC staining intensity, (2) does not depend on the precise dissection of the tissue on a gross scale and (3) considerably reduces the consequences of limited, arbitrary sampling of the region of interest for IHC analysis. The quantification procedure is illustrated by data from an experiment in which inflammatory reactions in the murine spinal cord, measured as microglial activation, were followed by IHC after the lesion of the sciatic nerve.

Effect of axotomy and 17ß-estradiol on P2X7 receptor expression pattern in the hypoglossal nucleus of ovariectomized mice.

The objective of the study was to examine whether axotomy and 17ß-estradiol affects P2X7 receptor expression and distribution in the hypoglossal nucleus. The left hypoglossal nerve of ovariectomized mice was cut and animals received a single injection of 17ß-estradiol (25 µg/100g b.w. in 20% (2-hydroxypropyl)-ß-cyclodextrin) or vehicle one hour after axotomy. Mice were sacrificed on day 4 following surgery. The area fraction of P2X7 receptor immunoreactive structures and of CD11b immunolabeled microglia, P2X7 protein concentration, and the immunoreactivity pattern of estrogen receptor (ER) alpha/beta were analyzed on both sides of the hypoglossal nucleus. Following axotomy the area fraction of P2X7 immunoreactive neurons showed a decreasing tendency, while the area fraction of P2X7 immunolabeled microglia increased significantly on the axotomized side compared with the control side in mice injected with vehicle. In animals treated with 17ß-estradiol the decrease in area fraction of neural and the increase in area fraction of microglial P2X7 immunostaining on the axotomized side were significantly enhanced compared with animals injected with vehicle. The P2X7 immunoreactivity pattern on the control side of the nucleus remained unchanged after 17ß-estradiol injection. Semi-quantitative Western blots revealed no significant difference in P2X7 protein concentration comparing the axotomized side with the control side in either experimental group. The CD11b immunoreactive microglia area fraction increased significantly following axotomy, but was not affected by 17ß-estradiol. Neither ER alpha, nor beta colocalized with CD11b. Our results suggest that axotomy induces cell-type specific changes in P2X7 receptor expression, which may be directly regulated by 17ß-estradiol through ER alpha or beta in neurons, but not in activated microglia.

Altered calcium homeostasis and ultrastructure in motoneurons of mice caused by passively transferred anti-motoneuronal IgG.

Calcium homeostasis and ultrastructure are altered in motor axon terminals (AT) of amyotrophic lateral sclerosis (ALS) patients and in mice injected with ALS IgG and exhibit increased density of synaptic vesicles and increased intracellular calcium. To develop an immune-mediated passive transfer experimental model of both systemic weakness and altered morphology, mice were inoculated intraperitoneally with anti-motoneuronal IgG. Animals initially manifested muscle stiffness and evidence of autonomic cholinergic hyperactivity. Electron microscopic cytochemistry within 12 hours (h) demonstrated significantly increased density of synaptic vesicles and calcium both in axon terminals of neuromuscular junctions and synaptic boutons on spinal motoneurons. After 24 h the mice were severely weak and premorbid. The number of synaptic vesicles was still larger than normal, but calcium was depleted from axon terminals and synaptic boutons. The motoneuron perikarya demonstrated the dilatation of the Golgi system and the rough endoplasmic reticulum with an increased amount of calcium. The NMDA receptor antagonist, MK-801, and the L-type calcium channel antagonist, Diltiazem, prevented clinical symptoms and some morphological alterations. These data demonstrate that high titer anti-motoneuronal IgG can induce severe weakness and produce similar ultrastructural features of motor axon terminals in human ALS and in mice injected with ALS IgG, and support a key role for calcium in selective vulnerability of motoneurons.

Intracellular calcium parallels motoneuron degeneration in SOD-1 mutant mice.

Transgenic mice with Cu,Zn superoxide dismutase (SOD-1) mutations provide a unique model to examine altered Ca homeostasis in selectively vulnerable and resistant motoneurons. In degenerating spinal motoneurons of G93 A SOD-1 mice, developing vacuoles were filled with calcium, while calcium was gradually depleted from the cytoplasm and intact mitochondria. In oculomotor neurons, no degenerative changes, vacuolization, or increased calcium were noted. Motor axon terminals of interosseus muscle gradually degenerated and intracellular calcium was depleted. Oculomotor terminals of mutant SOD-1 mice were smaller and exhibited no degenerative changes, but did exhibit unique membrane-enclosed organelles containing calcium. Spinal motoneurons of SOD-1 mice were shown to have fewer calcium binding proteins, such as parvalbumin, compared with oculomotor neurons. These data suggest that the SOD-1 mutation is associated with impaired calcium homeostasis in motoneurons in vivo, with increased likelihood of degeneration associated with higher levels of intracellular calcium and lower to absent levels of calbindin-D28K and/or parvalbumin, and decreased likelihood of degeneration associated with minimally changed calcium and ample calbindin-D28K and/or parvalbumin.

Neonatal enucleation induces correlated modification in sensory responsive areas and pial angioarchitecture of the parietal and occipital cortex of albino rats.

This study was carried out to investigate whether correlations existing in normal adult rats (Ambach et al., '86) between functional characteristics of neocortical areas and their pial angioarchitecture can be correspondingly modified under pathological conditions. The right eyes of albino rats were enucleated on the 1st, 8th, 15th and 30th day after birth, respectively. At the age of 3 to 4 months, the responsiveness of the parieto-occipital cortex to sensory stimuli was studied in enucleated animals and age matched controls. After the mapping of visually and somatosensorily evoked potentials, the vascular system was filled with dye. Monocular enucleation at birth induced bilateral modifications in sensory responsiveness and corresponding changes in pial angioarchitecture, especially in the venous drainage fields. In comparison with the controls, a considerable expansion was observed in the overlapping zone between visually and somatosensorily responsive areas. In contrast, borders of the visual cortex toward the auditory and retrosplenial areas were essentially stable. Corresponding changes were found in the pial distribution patterns of cerebral veins but not of arteries. The major effect of neonatal enucleation on angioarchitecture was a change in the subdivision of the parieto-occipital veins drainage fields. This was due to a significant enlargement of the anterior accessory occipital (O3) vein, which compressed the drainage fields of the parietal and occipital veins and completely separated them from one another. The results suggest that during ontogenesis: (1) alterations in the formation of sensory input may interfere with neocortical angiogenesis, especially the structuring of veins, (2) after monocular enucleation this influence is prominent in parietal and occipital cerebral veins, and (3) these angiogenetic processes are vulnerable only during the perinatal and early postnatal period.

Stereotaxic injection of IgG from patients with Alzheimer disease initiates injury of cholinergic neurons of the basal forebrain.

CONTEXT: The participation of an immune/inflammatory process in the pathomechanism of sporadic Alzheimer disease (AD) has been suggested by evidence for activated microglia and the potential therapeutic benefit of anti-inflammatory medication. OBJECTIVE: To define a possible role for IgG in the immune/inflammatory process of AD in humans, we assayed the ability of IgG samples from patients with AD to target the injury to cholinergic neurons in rat basal forebrain in vivo. DESIGN: IgG purified from the serum or plasma from patients with AD and patients with other neurological disease who were used as control (DC) patients was injected stereotaxically into the medial septum of adult rats. Four weeks later coronal sections of the whole medial septum-diagonal bands of Broca region were immunostained for choline acetyltransferase (ChAT) to identify cholinergic neuronal cells. SETTING: University medical centers. PATIENTS: Blood samples were collected from 8 patients with probable and definite AD and from 6 age-matched DC patients. MAIN OUTCOME MEASURE: Detection of changes in the number of ChAT immunopositive cell profiles in sections and statistical evaluation. RESULTS: Four weeks after the injections, IgG samples from patients with AD significantly reduced the number of ChAT-immunostained cell profiles in the whole medial septum-diagonal bands of Broca region compared with IgGs from DC patients. Neither DC IgGs nor saline solution significantly decreased the number of ChAT-immunopositive neuronal cell profiles. CONCLUSION: Data document that IgG from patients with AD can target a stereotaxically induced immune/inflammatory injury to cholinergic neurons in the rat basal forebrain in vivo.

Experimental destruction of substantia nigra initiated by Parkinson disease immunoglobulins.

BACKGROUND: Increased levels of free radicals and oxidative stress may contribute to the pathogenesis of substantia nigra (SN) injury in Parkinson disease (PD), but the initiating etiologic factors remain undefined in most cases. OBJECTIVE: To determine the potential importance of immune mechanisms in triggering or amplifying neuronal injury, we assayed serum samples from patients with PD to determine the ability of IgG to initiate relatively specific SN injury in vivo. METHODS: IgG purified from the serum of 5 patients with PD and 10 disease control (DC) patients was injected into the right side of the SN in adult rats. Coronal sections were cut from the whole brain at the level of the stereotaxic injections, stained for tyrosine hydroxylase and with cresyl violet, and cellular profiles were counted in identical brain regions at the injection and contralateral sides. The ratio of cell profile counts of the corresponding injected and uninjected regions was used as an internal standard. RESULTS: Four weeks following injection of IgG, a 50% decrease in tyrosine hydroxylase-positive cellular profiles was noted on the injected sides compared with the contralateral sides of the same animals. Similarly, applied DC IgG caused only an 18% decrease. Cresyl violet staining revealed a 35% decrease in neuronal profiles of PD IgG injected into the SN pars compacta compared with the contralateral uninjected side, whereas DC IgG caused a minimal 10% decrease. Even at 4 weeks after the PD IgG injections, perivascular inflammation and significant microglial infiltration were present near injured SN pars compacta neurons. No cytotoxic effects of PD IgG were noted in choline acetyltransferase-positive neurons after stereotaxic injections into the medial septal region. Absorption of PD IgG with mesencephalic membranes and protein A agarose gel beads removed cytotoxicity, while absorption with liver membranes did not change the cytotoxicity. CONCLUSIONS: Our data suggest that PD IgG can initiate a relatively specific inflammatory destruction of SN pars compacta neurons in vivo and demonstrate the potential relevance of immune mechanisms in PD.

Resistance of extraocular motoneuron terminals to effects of amyotrophic lateral sclerosis sera.

In sporadic ALS (s-ALS), axon terminals contain increased intracellular calcium. Passively transferred sera from patients with s-ALS increase intracellular calcium in spinal motoneuron terminals in vivo and enhance spontaneous transmitter release, a calcium-dependent process. In this study, passive transfer of s-ALS sera increased spontaneous release from spinal but not extraocular motoneuron terminals, suggesting that the resistance to physiologic abnormalities induced by s-ALS sera in mice parallels the resistance of extraocular motoneurons to dysfunction and degeneration in ALS.

Autoimmunity and ALS.

Significant evidence has accrued suggesting that antibodies to voltage-gated calcium channel are observed in at least some patients with sporadic ALS (SALS) and that such antibodies alter the function of these ion channels in vitro and in vivo. Further, passive transfer of these immunoglobulin-containing fractions into mice produces changes at the neuromuscular junction that are very similar to changes observed in patients with SALS. These changes reflect local alterations in intracellular Ca2+ homeostasis and, in animal models, may also evidence early changes of motoneuron injury, such as Golgi apparatus swelling and fragmentation. Although not yet documented to induce motoneuron death in vivo, SALS immunoglobulins induce Ca(2+)-dependent apoptosis in a differentiated motoneuron hybrid cell line via a mechanism that involves oxidative injury. SALS immunoglobulin-mediated apoptosis in these cells is regulated by the presence of the same calcium-binding proteins that may modulate selective motoneuron vulnerability in SALS.

Immunization of guinea pigs with human choline acetyltransferase induces selective lower motoneuron destruction.

Prior studies have demonstrated that guinea pigs immunized with bovine spinal motoneurons develop immune-mediated lower motoneuron disease. In the present experiments, guinea pigs immunized with choline acetyltransferase (ChAT) from human placenta develop lower motoneuron destruction and striated muscle atrophy. In this model, increased IgG was detected in lower motoneurons and at the motor end-plate by immunocytochemistry. Ultrastructural analysis revealed an increase in calcium content and in the density of synaptic vesicles in axon terminals at neuromuscular junctions. Similar morphological changes could be induced in mice following passively transfer of IgG from ChAT-immunized guinea pigs. The increased IgG uptake and raised calcium content in motor axon terminals as well as the selective lower motoneuron damage, suggest that a similar final common pathway can lead to motoneuron injury following immunization with human placental ChAT or bovine spinal motoneurons.

Intracellular calcium redistribution accompanies changes in total tissue Na+, K+ and water during the first two hours of in vitro incubation of hippocampal slices.

Changes of total tissue water, Ca, Na and K contents were monitored in whole transverse hippocampal slices of the guinea-pig during the first 2 h of in vitro incubation. A brief, 75% increase in tissue Ca was noted during the initial 15 min of maintenance, in contrast to a permanent increase of sodium and water contents, coupled to simultaneous decrease of potassium level. The rate of tissue Na, K and water changes comprised a rapid phase at the first 10-20 min, parallel with the increase of the tissue Ca content, and a slow phase during the rest of the incubation period. Development of specific morphological alterations, representative of ischemic/hypoxic lesions and a translocation of calcium from cytoplasm to mitochondria and endoplasmic reticulum during slice maintenance, was also detected by electron microscopy. A two-step mechanism might explain the development of a new steady-state total calcium content of slices. in which the cellular Ca2+ uptake at the beginning of incubation, likely triggered by hypoxic/ ischemic trauma of slice preparation, is followed by a balanced Ca2+ influx, extrusion and sequestration (predominantly into mitochondria and endoplasmic reticulum) during maintenance.

Transient increase of calcium in pre- and postsynaptic organelles of rat superior cervical ganglion after tetanizing stimulation.

The stimulation-dependent translocation and redistribution of intracellular calcium was studied in synapses of the rat superior cervical ganglion. For the cytochemical demonstration of calcium at the ultrastructural level the oxalate-pyroantimonate technique was used. Calcium-containing deposits were observed as electron-dense particles in synaptic vesicles and occasionally in presynaptic mitochondria. On the postsynaptic side, vesicles and vacuoles in dendrites contained increased amounts of reaction product. Following a short train of impulses (20 s, 20 c.p.s.), the ganglionic response to a single stimulus was increased. This potentiation effect which lasted about 1-5 min was accompanied by a change in the distribution of calcium at fine structural level. In ganglia fixed 1 and 5 min after the train, but not later, the number of calcium-containing synaptic vesicles and postsynaptic vacuoles increased significantly.

Fine structural changes in the superior cervical ganglion of adult rats after long-term administration of baclofen, a GABAB receptor agonist.

The aim of the present study was to examine the fine structural changes in the superior cervical ganglion of adult rats following baclofen treatment. On the addition of this GABAB receptor agonist there was a transient increase in the number of coated vesicles and pits in the dendrites of principal sympathetic neurons. As compared to the control ganglia, we found a significantly higher number of synapses which had coated pits attached to the postsynaptic densities. In interpreting these observations it is suggested that the GABAB receptors play some role in mediating the synaptogenetic action of GABA, although the formation of vacant postsynaptic densities and spines was not seen after baclofen treatment.

Chloride is preferentially accumulated in a subpopulation of dendrites and periglomerular cells of the main olfactory bulb in adult rats.

GABA is predominantly an inhibitory transmitter. Mediated by GABAA receptors, GABA opens chloride channels, induces a passive flux of chloride ions, which is usually directed from extracellular to intracellular space, and hyperpolarizes postsynaptic neurons. Recent electrophysiological data suggested that GABA may also depolarize neurons and exert excitatory actions. However, it remained unclear whether excitatory GABA effects are based on reversed transmembrane chloride gradient due to modifications in extracellular or intracellular chloride concentrations. Here, the first histochemical evidence is provided for local redistribution of chloride in the CNS of healthy adult rats. Olfactory bulbs were examined using freeze substitution, silver trapping of chloride and intensification techniques at light and electron microscopic level. The chloride content of precipitates was evidenced by electron spectroscopic imaging using a CEM 902 (Zeiss) electron microscope. Chloride concentration was high in a subpopulation of some periglomerular cell bodies and isolated dendritic profiles, while it seemed to be very low in certain parts of the glomerular neuropil including intercellular clefts. Data suggest that reversed chloride gradients can be demonstrated by cytochemical methods, and may be responsible for excitatory GABA effects on selected periglomerular neurons and dendrites in the olfactory glomeruli. Conditions leading to chloride redistribution in the CNS of normal adult rats remain to be determined.

Calcium-containing endosomes at oculomotor terminals in animal models of ALS.

Altered calcium homeostasis has been demonstrated in human spinal cord motor axon terminals of ALS patients, in spinal motor neurons of mutant SOD transgenic mice and following injection of ALS immunoglobulins. In all three paradigms oculomotor neurons are relatively spared. To explore mechanisms of selective resistance, we applied similar calcium localization techniques to terminals of oculomotor neurons in the two animal models. In both cases large vacuoles, which connect with the extracellular space, accumulated the majority of intracellular calcium, while terminals of vulnerable neurons (e.g. innervating interosseus muscle), which possess no such vacuoles, displayed evenly distributed calcium. These relatively unique membrane enveloped structures may permit neurons to control their cytoplasmic Ca2+ concentration and contribute to selective resistance.

Recruitment of activated microglia cells in the spinal cord of mice by ALS IgG.

Mice were injected i.p. with IgG samples of different patients to test whether IgG from amyotrophic lateral sclerosis (ALS) can initiate an immune/inflammatory reaction targeting motor neurons. All IgG samples of five ALS patients and none of the disease controls recruited activated microglia cells in the ventral horn of the spinal cord. CD3 lymphocytes were not accumulated in the same tissue. Similar reaction was evoked by injection of IgG from guinea pigs with experimental autoimmune gray matter disease (EAGMD) induced by immunization with the homogenate of the ventral horn of bovine spinal cord. The results indicate that ALS IgG and anti-motoneuron IgG induce microglia reaction targeting motor neurons without initiating T cell response in the recipient mice.

Algorithm for semi-automatic sorting of objects to specified tissue domains. An aid for co-ordinating morphometric data with identified tissue components.

A personal computer-based technique was developed that reduces the extent of human efforts in obtaining data, like numerical density of profiles on area, describing the distribution of specific features of histological sections, e.g., density of synaptic profiles or of histochemical reaction products in specified tissue compartments. The procedure consists of (i) marking the objects to be counted, (ii) recording the borderlines of (reference) tissue domains of interest, and (iii) allocating the marked objects to the corresponding domains automatically. This automatic sorting of objects into defined tissue domains is achieved by an algorithm operating on two sets of coordinates: (i) coordinates of points that constitute the boundary of tissue domains (perimeter points) and (ii) coordinates of points marking the particular objects to be sorted. The principle of the sorting calculation is to construct 'segments' of the loop by lines parallel to the ordinate which pass through neighbouring perimeter points. The 'items' to be sorted are classified by the sign of marker flags allocated to each point depending on which side of the perimeter segment they are located. Segmentation and classification procedures are sequentially repeated along the entire perimeter of each domain specified by the operator which may result during the procedure in multiple changes of the sign of the flags. The internal or external location of each item finally is represented by the last sign of its flag. Objects, allocated to domains can be counted and processed further for numerical density determination.

Semiquantitative evaluation of histochemically detectable calcium binding in mammalian brain slices.

A method is described for the electron histochemical demonstration of Ca in the central nervous system, based on fixation on Ca-containing paraformaldehyde solution and the subsequent complexing of Ca by ammonium oxalate. The method resulted in highly electron-dense deposits, with good ultrastructural preservation. The Ca content of the deposits was proved by physico-chemical analysis. The high electron density permitted the counting of deposits and thereby an estimation of their numerical density, via planimetry of electron micrographs. Since pre- and postsynaptic localizations could be distinguished on the basis of ultrastructure, this procedure is regarded as a unique semiquantitative method for estimation of the tissue Ca binding of mammalian brain slices.

A new cytochemical method for the ultrastructural localization of calcium in the central nervous system.

We have developed a new cytochemical method for the localization of calcium at the ultrastructural level in the central nervous system (CNS). The method is based on the use of phosphate buffer in the primary fixation followed by a mixture of a complex of chromium(III)-trisoxalate and osmium tetroxide (OsO4) which precipitates calcium and results in the formation of a high electron-dense reaction product. Calcium selectivity was verified by reactions made in test tube, by EGTA treatment of the tissue, by electron spectroscopic imaging (ESI) and electron energy loss spectroscopy (EELS). The technique was found to be reproducible, yielding similar results in acutely prepared hippocampal slices or organotypic cultures fixed by immersion and in brain areas fixed by perfusion. In hippocampal slices, calcium deposits were found to accumulate in different subcellular compartments such as endoplasmic reticulum, mitochondria and synaptic vesicles. Interestingly, electron-dense reaction products were also visualized in smooth endoplasmic reticulum structures localized in presynaptic terminals or post-synaptic spines as well as in synaptic clefts and active zones. This new method may thus be of interest for studying the metabolism of calcium, specifically with regard to synaptic activity, in the CNS.

Calcium accumulation by dendritic mitochondria declines along the apical dendrites of pyramidal neurons in area CA1 of guinea pig hippocampal slices.

An electron microscopic histochemical study was performed in stratum radiatum of area CA1 of guinea pig hippocampal slices in order to determine the spatial distribution of a dendritic mitochondrial subpopulation which accumulated calcium during in vitro incubation. A distribution gradient was found along the course of apical dendrites exhibiting the highest density values at the base of the dendrites and decaying to baseline values at about 50 microns distal from the cell body layer. The pronounced calcium accumulation by mitochondria in the proximal apical dendrites was markedly but not completely reduced by blocking L-type Ca-channels. These results (i) support the observation of a clustered distribution of L-type Ca-channels at the base of apical dendrites, (ii) designate these voltage dependent Ca2+ channels as one of the possible routes for calcium influx caused by hypoxia/ischemia induced during slice preparation, and (iii) emphasize the role of mitochondrial calcium sequestering under ischemic/hypoxic conditions.