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.

Hematopoietic stem cell transplantation in patients with sporadic amyotrophic lateral sclerosis.

BACKGROUND: Amyotrophic lateral sclerosis (ALS), an inexorably progressive motoneuron disease, is accompanied by significantly increased markers of inflammation. These inflammatory constituents could protect, harm, do neither, or do both. OBJECTIVE: Allogeneic hematopoietic stem cell transplantation (HSCT) was performed in patients with sporadic ALS to suppress neuroinflammation and improve clinical outcomes after CNS engraftment. METHODS: Six patients with definite ALS received total body irradiation followed by peripheral blood HSCT infusion from human leukocyte antigen identically matched sibling donors. Disease progression and survival were assessed monthly and compared with matched historic database patients. Autopsy samples from brain and spinal cord were examined immunohistochemically and by quantitative reverse-transcriptase polymerase chain reaction. Donor-derived DNA in brain and spinal cord tissue was evaluated for the extent of chimerism. RESULTS: No clinical benefits were evident. Four patients were 100% engrafted; postmortem tissue examination in two of the 100% engrafted patients demonstrated 16% to 38% donor-derived DNA at sites with motoneuron pathology, which may correspond to the observed increased CD68 or CD1a-positive cells. Neither donor DNA nor increased cell numbers were found in several unaffected brain regions. A third minimally engrafted patient had neither donor DNA nor increased infiltrating cells in the CNS. CONCLUSIONS: This study demonstrates that peripheral cells derived from donor hematopoietic stem cells can enter the human CNS primarily at sites of motoneuron pathology and engraft as immunomodulatory cells. Although unmodified hematopoietic stem cells did not benefit these sporadic amyotrophic lateral sclerosis patients, such cells may provide a cellular vehicle for future CNS gene therapy.

Subcellular localization of IgG from the sera of ALS patients in the nervous system.

Immunoglobulin G (IgG) samples isolated from the sera of amyotrophic lateral sclerosis (ALS) and control patients were injected intraperitoneally into mice. After 24 h the mice were processed for immune electron microscopic immunohistochemistry to localize IgG in their nervous system. The injected ALS IgG was observed in the axon terminals of the lower motor neurons (MNs), localized to the microtubules and enriched in the rough endoplasmic reticulum (RER). In post-mortem spinal cord samples from ALS patients, IgG was similarly detected in the vicinity of the microtubules and in the RER of the MNs. IgG was neither found in the corresponding structures of MNs of mice injected with the control human IgG nor in post-mortem human control spinal cord samples. The data suggest that multiple antibodies directing to different structures of the MNs may play a role in their degeneration in ALS.

Widespread increased expression of the DNA repair enzyme PARP in brain in ALS.

Expression of the DNA repair enzyme poly(ADP-ribose) polymerase (PARP) is a known response to oxidative damage of DNA. In ALS brain, PARP expression by western analyses was increased in the motor cortex, parietal cortex, and cerebellum. PARP immunostaining in the motor cortex was increased in ALS neurons and subcortical glia and macrophages. Importantly, there was widespread increased PARP expression in neurons in the parietal cortex and cerebellum, regions that are typically clinically unaffected in ALS, suggesting widespread oxidative stress.

Altered calcium in motoneurons by IgG from human motoneuron diseases.

OBJECTIVES: The effect of IgG from patients with multifocal motor neuropathy (MMN) on the content and distribution of calcium in spinal motoneurons was compared with the effect of IgG from patients with sporadic amyotrophic lateral sclerosis (SALS) and IgG from normal individuals. MATERIAL AND METHODS: Different purified IgG samples were injected intraperitoneally in mice. Then, the animals were subjected to histochemical techniques to visualize calcium in electron microscopic sections. RESULTS: Quantitative morphometric analysis verified that IgG from MMN decreased the vesicular and axoplasmic calcium content in the axon terminals at the neuromuscular junctions and had no influence on the perikaryon. In contrast to this, IgG from patients with SALS increased the intracellular calcium both in the axon terminal and in the perikaryon. IgG from normal individuals exerted no effect. Elevated intracellular calcium may contribute to motoneuron degeneration. The lack of such effect with MMN immunoglobulins helps to explain the relative sparing of motoneurons in the disease.

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.

Calcium: the Darth Vader of ALS.

Motor neuron dysfunction and loss in amyotrophic lateral sclerosis (ALS) have been attributed to several different mechanisms, including increased intracellular calcium, glutamate excitotoxicity, oxidative stress and free radical damage, mitochondrial dysfunction, and neurofilament aggregation and dysfunction of transport mechanisms. These alterations are not mutually exclusive, and increased calcium could be a common denominator. Furthermore, the selective vulnerability of spinal motor neurons and the relative sparing of eye motor neurons represent striking features of both sporadic and familial ALS. Here we review the evidence that calcium homeostasis is altered in ALS, and that low levels of the calcium binding proteins parvalbumin and calbindin-D28K contribute to selective vulnerability by decreasing the ability of motor neurons to handle an increased calcium load, with cell injury and death as the consequence.

UVB irradiation-induced apoptosis increased in lymphocytes of Huntington's disease patients.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by CAG repeat expansion in the IT-15 gene coding for huntingtin. The mechanism of neuronal degeneration induced by the mutant huntingtin is not known. Apoptosis may play a role in it. Huntingtin is widely expressed in the cells, so abnormalities can be expected also in non-neural tissue. We examined the susceptibility of lymphocytes from HD patients, asymptomatic carriers and normal individuals to UVB irradiation-induced apoptosis. Lymphocytes from eight HD patients and two asymptomatic carriers showed increased apoptotic cell death compared to controls. Our results suggests that sensitivity of HD cells to induced apoptosis is not restricted to neurons.

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.

Altered calcium homeostasis in spinal motoneurons but not in oculomotor neurons of SOD-1 knockout mice.

SOD-1-deficient mice demonstrate no loss of motoneurons but are still vulnerable to axotomy and ischemic insults. To investigate possible reasons for vulnerability of motoneuron populations, we studied changes in ultrastructural calcium distribution during maturation in spinal- and oculomotor neurons in SOD-1(-/-) mice. Between 3 and 11 months the cytoplasmic component of the intracellular calcium changed at a lower rate in spinal motoneurons and motor axon terminals in the interosseus muscle of SOD-1(-/-) animals compared to wild-type controls. No such dissimilarities were noted in the oculomotor system, or in mitochondrial calcium contents of either cell type. These data suggest that the lack of SOD-1 may be associated with vulnerability to insult by depletion of non-mitochondrial calcium stores selectively in motoneurons lacking parvalbumin and/or calbindin D28K.

Ultrastructural evidence of calcium involvement in experimental autoimmune gray matter disease.

Experimental studies have suggested that increased calcium and inappropriate calcium handling by motoneurons might have a significant role in motoneuron degeneration. To further define the involvement of calcium in motoneuron loss we used the oxalate-pyroantimonate technique for calcium fixation and monitored the ultrastructural distribution of calcium in spinal motoneurons in experimental autoimmune gray matter disease (EAGMD). In cervical and hypoglossal motoneurons from animals with relatively preserved upper extremity and bulbar function, increased calcium precipitates were present in the cytoplasm as well as in mitochondria, endoplasmic reticulum and Golgi complex without significant morphologic alterations. In surviving lumbar motoneurons of animals with hindlimb paralysis, however, there was massive morphological destruction of intracellular organelles but no significant accumulation of calcium precipitates. These findings suggest that altered calcium homeostasis is involved in motoneuron immune-mediated injury with increased calcium precipitates early in the disease process and decreased to absent calcium precipitates later in the pathogenesis of motoneuron injury.

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.

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.

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.

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.

Experimental immune-mediated damage of septal cholinergic neurons.

Degeneration of cholinergic neurons in the medial septum and the diagonal band of Broca is a frequent neuropathological feature of Alzheimer's disease. To determine whether an immune process can injure these basal forebrain cholinergic neurons, we serially immunized guinea pigs with septal cholinergic hybrid cells (SN-56). Following immunization, a relatively selective damage of septal cholinergic neurons, reduction in septal choline acetyltransferase (ChAT) activity and decrease in acetylcholine release in hippocampus were detected. Serum IgG from guinea pigs immunized with SN-56 cells and stereotactically injected into the medial septal region of rats produced a loss of ChAT activity in the medial septum, frontal cortex and hippocampus, together with impairment of learning and long term spatial memory. These data suggest that relatively selective damage to septal cholinergic neurons can be caused by an immune-mediated process in experimental animals.

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.

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.

Antibodies to calcium channels from ALS patients passively transferred to mice selectively increase intracellular calcium and induce ultrastructural changes in motoneurons.

Antibodies to Ca channels in ALS patients IgG can be demonstrated to enhance Ca current and cause cell injury and death in a motoneuron cell line in vitro. To determine whether these antibodies can alter neuronal calcium homeostasis in vivo IgG fractions from six ALS patients were injected intraperitoneally into mice, and neurons assayed by ultrastructural techniques for calcium content. After 24 h, all six ALS IgG by (40 mg/animal) increased vesicle number in spinal motoneuron axon terminals, and in boutons synapsing on spinal motoneurons. Using the oxalate-pyroantimonate technique for calcium precipitation, these antibodies produced dose-dependent calcium increases either in axon terminal synaptic vesicles and mitochondria, or in rough endoplasmic reticulum, mitochondria, and Golgi complex of spinal motoneuron and frontal cortex pyramidal cells. ALS IgG was itself internalized and also induced neurofilament H phosphorylation. The observed changes in ultrastructure and calcium compartmentation were restricted to motoneurons; normal and disease control IgG, which did not possess antibodies enhancing calcium entry, did not exert similar effects. These data demonstrate that ALS IgG containing Ca-channel antibodies can alter calcium homeostasis of motoneurons in vivo.