Advanced Stages Of Degeneration Research Articles

Targeted screening of inflammatory mediators in spontaneous degenerative disc disease in dogs reveals an upregulation of the tumor necrosis superfamily.

The regulation of inflammatory mediators in the degenerating intervertebral disc (IVD) and corresponding ligamentum flavum (LF) is a topic of emerging interest. The study aimed to investigate the expression of a broad array of inflammatory mediators in the degenerated LF and IVD using a dog model of spontaneous degenerative disc disease (DDD) to determine potential treatment targets. LF and IVD tissues were collected from 22 normal dogs (Pfirrmann grades I and II) and 18 dogs affected by DDD (Pfirrmann grades III and IV). A qPCR gene array was used to investigate the expression of 80 inflammatory genes for LF and IVD tissues, whereafter targets of interest were investigated in additional tissue samples using qPCR, western blot (WB), and immunohistochemistry. Tumor necrosis factor superfamily (TNFSF) signaling was identified as a regulated pathway in DDD, based on the significant regulation (n-fold ± SD) of various TNFSF members in the degenerated IVD, including nerve growth factor (NGF; -8 ± 10), CD40LG (464 ± 442), CD70 (341 ± 336), TNFSF Ligand 10 (9 ± 8), and RANKL/TNFSF Ligand 11 (85 ± 74). In contrast, TNFSF genes were not significantly affected in the degenerated LF compared to the control LF. Protein expression of NGF (WB) was significantly upregulated in both the degenerated LF (4.4 ± 0.5) and IVD (11.3 ± 5.6) compared to the control group. RANKL immunopositivity was significantly upregulated in advanced stages of degeneration (Thompson grades IV and V) in the nucleus pulposus and annulus fibrosus of the IVD, but not in the LF. DDD involves a significant upregulation of various TNFSF members, with tissue-specific expression profiles in LF and IVD tissues. The differential involvement of TNFSF members within multiple spinal tissues from the same individual provides new insights into the inflammatory processes involved in DDD and may provide a basis to formulate hypotheses for the determination of potential treatment targets.

Expression of Glial-Cell-Line-Derived Neurotrophic Factor Family Ligands in Human Intervertebral Discs.

Glial-cell-line-derived neurotrophic factor (GDNF) family ligands (GFLs) contribute to the sensitization of primary afferents and are involved in the pathogenesis of inflammatory pain. The purpose of this preliminary study was to examine the expression of other GFLs (neurturin (NRTN), artemin (ARTN), persephin (PSPN)) and receptors in human IVD cells and tissues exhibiting early and advanced stages of degeneration. Human IVD cells were cultured as a monolayer after isolation from the nucleus pulposus (NP) and anulus fibrosus (AF) tissues. The mRNA expression of NRTN, ARTN, PSPN, and their receptors (GFRA2-GFRA4) was quantified using real-time PCR. Protein expression was evaluated using immunohistochemistry and Western blotting. The expression of NRTN, ARTN, PSPN, and their co-receptors (GFRA2-GFRA4) was identified in human IVD cells at both mRNA and protein levels. A trend was noted wherein the mRNA expression of ARTN, PSPN, and GFRA2 was upregulated by IL-1β treatment in a dose-dependent manner. The percentages of immunopositive cells in the advanced degenerate stage of ARTN, PSPN, and GFRA2 were significantly higher than those in the early degenerate stage. Their expression was enhanced in advanced tissue degeneration, which suggests that GFLs (ARTN and PSPN) may be involved in the pathogenesis of discogenic pain.

Expression of GADD45G and CAPRIN1 in Human Nucleus Pulposus: Implications for Intervertebral Disc Degeneration.

Marked cellular changes occur in human intervertebral disc (IVD) degeneration during disc degeneration with biochemical changes. Genome-wide analysis of the DNA methylation profile has identified 220 differentially methylated loci associated with human IVD degeneration. Among these, two cell-cycle-associated genes, growth arrest and DNA damage 45 gamma (GADD45G) and cytoplasmic activation/proliferation-associated protein-1 (CAPRIN1), were focused on. The expression of GADD45G and CAPRIN1 in human IVDs remains unknown. We aimed to examine the expression of GADD45G and CAPRIN1 in human nucleus pulposus (NP) cells and evaluate those in human NP tissues in the early and advanced stages of degeneration according to Pfirrmann magnetic resonance imaging (MRI) and histological classifications. Human NP cells were cultured as monolayers after isolation from NP tissues by sequential enzyme digestion. Total RNA was isolated, and the mRNA expression of GADD45G and CAPRIN1 was quantified using real-time polymerase chain reaction. To examine the effects of pro-inflammatory cytokines on mRNA expression, human NP cells were cultured in the presence of IL-1β. Protein expression was evaluated using Western blotting and immunohistochemistry. GADD45G and CAPRIN1 expression was identified in human NP cells at both mRNA and protein levels. The percentage of cells immunopositive for GADD45G and CAPRIN1 significantly increased according to the Pfirrmann grade. A significant correlation between the histological degeneration score and the percentage of GADD45G-immunopositive cells was identified, but not with that of CAPRIN1-immunopositive cells. The expression of cell-cycle-associated proteins (GADD45G and CAPRIN1) was enhanced in human NP cells at an advanced stage of degeneration, suggesting that it may be regulated during the progression of IVD degeneration to maintain the integrity of human NP tissues by controlling cell proliferation and apoptosis under epigenetic alteration.

Quantitative characterisation of ipRGCs in retinal degeneration using a computation platform for extracting and reconstructing single neurons in 3D from a multi-colour labeled population.

Light has a profound impact on mammalian physiology and behavior. Intrinsically photosensitive retinal ganglion cells (ipRGCs) express the photopigment melanopsin, rendering them sensitive to light, and are involved in both image-forming vision and non-image forming responses to light such as circadian photo-entrainment and the pupillary light reflex. Following outer photoreceptor degeneration, the death of rod and cone photoreceptors results in global re-modeling of the remnant neural retina. Although ipRGCs can continue signaling light information to the brain even in advanced stages of degeneration, it is unknown if all six morphologically distinct subtypes survive, or how their dendritic architecture may be affected. To answer these questions, we generated a computational platform-BRIAN (Brainbow Analysis of individual Neurons) to analyze Brainbow labeled tissues by allowing objective identification of voxels clusters in Principal Component Space, and their subsequent extraction to produce 3D images of single neurons suitable for analysis with existing tracing technology. We show that BRIAN can efficiently recreate single neurons or individual axonal projections from densely labeled tissue with sufficient anatomical resolution for subtype quantitative classification. We apply this tool to generate quantitative morphological information about ipRGCs in the degenerate retina including soma size, dendritic field size, dendritic complexity, and stratification. Using this information, we were able to identify cells whose characteristics match those reported for all six defined subtypes of ipRGC in the wildtype mouse retina (M1-M6), including the rare and complex M3 and M6 subtypes. This indicates that ipRGCs survive outer retinal degeneration with broadly normal morphology. We additionally describe one cell in the degenerate retina which matches the description of the Gigantic M1 cell in Humans which has not been previously identified in rodent.

P.61 Imaging Mass Cytometry reveals new clues to understand the pathogenesis of Becker muscular dystrophy

Muscular dystrophies are genetic diseases characterised by muscle fiber loss and its replacement by fibrotic and adipose tissue. Several cell types, including macrophages and fibroadipogenic precursor cells (FAPS) interact with degenerative muscle fibers locally in what is known as the degenerative niche. Recent technological advances known as spatial biology allow dissecting cell-to-cell interactions in vivo using samples of tissue, although this has not been applied to human muscle tissue yet. We analysed muscle biopsies of controls and patients with Becker muscular dystrophy (BMD) at early, moderate and advanced stages of degeneration using Hyperion Imaging Mass Cytometry (IMC). We labelled single sections of the biopsies with 40 markers labelling different components of the muscle structure. We developed a deep learning algorithm to analyse the images and studied changes in the composition and spatial correlations of the different markers across disease progression. We included 2 control and 8 muscle biopsies of BMD. We observed a progressive increase in the amount of collagen-I and III, that significantly correlated with the amount of CD68+ macrophages (R=0.93 and R=0.94), most of them positive for the M2 marker CD206. Fat and collagen I surrounded vessels during the first stages of disease progression, substituting muscle fibres in later stages. We observed a negative correlation between regenerative muscle fibers and collagen-I (R=-0.83) and with CD68 cells (R:-0.90). We also observed a modification in the number of satellite cells in the biopsies across disease progression. IMC enables the study of changes in muscle structure along disease progression in patients with muscular dystrophies establishing spatio-temporal correlations opening the door to confirm and understand new potential pathogenic pathways in vivo in human samples.

The P2X7 purinergic receptor in intervertebral disc degeneration.

Mechanisms involved in the development of intervertebral disc (IVD) degeneration are only partially known, thus making the implementation of effective therapies very difficult. In this study, we investigated P2X7 purinergic receptor (P2X7R), NLRP3 inflammasome, and interleukin (IL)‐1β expression in IVD specimens at different stages of disease progression, and during the in vitro dedifferentiation process of the primary cells derived thereof. We found that P2X7R, NLRP3, and IL‐1β expression was higher in the IVD samples at a more advanced stage of degeneration and in the expanded IVD cells in culture which partially recapitulated the in vivo degeneration process. In IVD cells, the P2X7R showed a striking nuclear localization, while NLRP3 was mainly cytoplasmic. Stimulation with the semiselective P2X7R agonist benzoyl ATP together with lipopolysaccharide treatment triggered P2X7R transfer to the cytoplasm and P2X7R/NLRP3 colocalization. Taken together, these findings support pathophysiological evidence that the degenerated disc is a highly inflamed microenvironment and highlight the P2X7R/NLRP3 axis as a suitable therapeutic target. The immunohistochemical analysis and the assessment of subcellular localization revealed a substantial expression of P2X7R also in normal disc tissue. This gives us the opportunity to contribute to the few studies performed in natively expressed human P2X7R so far, and to understand the possible physiological ATP‐mediated P2X7R homeostasis signaling. Therefore, collectively, our findings may offer a new perspective and pave the way for the exploration of a role of P2X7R‐mediated purinergic signaling in IVD metabolism that goes beyond its involvement in inflammation.

Expression of Glial Cell Line-derived Neurotrophic Factor in the Human Intervertebral Disc.

Biochemical and immunohistochemical analyses by the human intervertebral disc (IVD) cells and tissues. To examine the expression of glial cell line-derived neurotrophic factor (GDNF) and its receptors, GDNF family receptor (GFR) α1 and rearranged during transfection (RET) in the human IVD cells and the tissues with the early and advanced stages of degeneration. The neurotrophin family, including nerve growth factor, has been reported to be expressed in the IVDs and plays a role in hyperalgesia and neuronal sensitization. Despite having properties similar to the nerve growth factor, the expression of GDNF in the IVD remains unknown. Human IVD cells were cultured in monolayer. Immunohistochemical analyses and western blotting were performed to examine the protein levels of GDNF and its receptors. To examine the effect of proinflammatory cytokines, cells were cultured in the presence of interleukin-1β (IL-1β). The immunohistochemical expression of these proteins was also evaluated using human IVD tissues with different stages of degeneration. Immunofluorescent reactivity against anti-GDNF, GFRα1, and RET antibodies was identified in human IVD cells. In protein extracts from IVD cells, those protein expressions were also identified by Western blot. IL-1β significantly stimulated the mRNA expression of GDNF compared with that of the control group. There was no significant effect of IL-1β on the mRNA expression of GFRα1 and RET. The percentage of GDNF-immunopositive cells in advanced degenerated discs was significantly higher than that in early degenerated discs, whereas those of GFRα1 and RET showed no significant differences. GDNF and its receptors were constitutively expressed in the human IVD cells. GDNF expression was significantly enhanced by proinflammatory stimuli, and in the microenvironment with advanced tissue degeneration. N/A.

Genomic Loss and Silencing on the Y Chromosomes of Rumex.

Across many unrelated lineages of plants and animals, Y chromosomes show a recurrent pattern of gene degeneration and loss, but the relative importance of inefficient selection, adaptive gene silencing, and neutral genetic drift in causing degeneration remain poorly understood. Here, we use next-generation genome and transcriptome sequencing to investigate patterns of ongoing Y chromosome degeneration in two annual plant species of Rumex (Polygonaceae) differing in their degree of degeneration and sex chromosome heteromorphism. We find evidence for both gene loss as well as silencing in these young plant sex chromosomes. Our analyses revealed significantly more gene deletion relative to silencing in R. rothschildianus, which has had a larger nonrecombining region for a longer period than R. hastatulus, consistent with this system being at a more advanced stage of degeneration. Intra- and interspecific comparisons of genomic coverage and heterozygosity indicated that loss of expression precedes gene deletion, implying that the final stages of mutation accumulation and gene loss may often occur neutrally. We found no evidence for adaptive silencing of genes that have lost expression. Our results suggest that the initial spread of deleterious regulatory variants and/or epigenetic silencing may be an important driver of early degeneration of Y chromosomes.

DNA damage precedes apoptosis during the regression of the interdigital tissue in vertebrate embryos.

DNA damage independent of caspase activation accompanies programmed cell death in different vertebrate embryonic organs. We analyzed the significance of DNA damage during the regression of the interdigital tissue, which sculpts the digits in the embryonic limb. Interdigit remodeling involves oxidative stress, massive apoptosis and cell senescence. Phosphorylation of H2AX mediated by ATM precedes caspase dependent apoptosis and cell senescence during interdigit regression. The association of γH2AX with other downstream DNA repair factors, including MDC1, Rad50 and 53BP1 suggests a defensive response of cells against DNA damage. The relative distribution of cells γH2AX-only positive, TUNEL-only positive, and cells double positive for both markers is consistent with a sequence of degenerative events starting by damage of the DNA. In support of this interpretation, the relative number of γH2AX-only cells increases after caspase inhibition while the relative number of TUNEL-only cells increases after inhibition of ATM. Furthermore, cultured interdigits survived and maintained intense chondrogenic potential, even at advanced stages of degeneration, discarding a previous commitment to die. Our findings support a new biological paradigm considering embryonic cell death secondary to genotoxic stimuli, challenging the idea that considers physiological cell death a cell suicide regulated by an internal death clock that pre-programmes degeneration.

Early disintegration of myonuclear fibrous lamina in inclusion body myositis: Ultrastructural and immunohistochemical studies

AbstractObjectiveMorphological analyses to assess the integrity of myonuclear surface structures in muscle biopsies from patients with inclusion body myositis (IBM) and related disorders were carried out to find clues to the causes of IBM.MethodsMuscle biopsies from 26 patients (seven with IBM, five polymyositis, three dermatomyositis, one hereditary inclusion body myopathy associated with Paget disease and front‐temporal dementia, three with other non‐inflammatory myopathies, four with no pathological abnormalities and three type 2 fiber atrophy only) were assessed by measuring lengths of clearly identifiable muscle outer (ONM) and inner nuclear membranes (INM), and fibrous laminae (FL) on electron micrographs, and grading severity of degenerative changes in the sarcoplasm into three stages. The nuclei of capillary endothelial cells were also examined and lamin A/C, a major constituent of FL, localized immunohistochemically.ResultsCompared with patients with no pathology or type 2 fiber atrophy only, polymyositis had significantly less ONM, INM and FL at an advanced stage of degeneration. In dermatomyositis, ONM was moderately decreased. In IBM, ONM, INM and FL were decreased severely from early stages of muscle fiber degeneration. In the one case with inclusion body myopathy associated with Paget disease and front‐temporal dementia, all three layers, particularly ONM, were decreased. No significant changes were observed in the nuclei of capillary endothelial cells in IBM or other conditions. In IBM, there was less lamin A/C in myonuclei than in capillary endothelial cell nuclei.ConclusionsIn IBM, shorter lengths of myonuclear surface structures, particularly of FL, were observed from an early stage of degeneration. Early disintegration of FL might reflect an essential pathomechanism of IBM.

Three-dimensional morphological and signal intensity features for detection of intervertebral disc degeneration from magnetic resonance images

Advances in MRI hardware and sequences are continually increasing the amount and complexity of data such as those generated in high-resolution three-dimensional (3D) scanning of the spine. Efficient informatics tools offer considerable opportunities for research and clinically based analyses of magnetic resonance studies. In this work, we present and validate a suite of informatics tools for automated detection of degenerative changes in lumbar intervertebral discs (IVD) from both 3D isotropic and routine two-dimensional (2D) clinical T2-weighted MRI. An automated segmentation approach was used to extract morphological (traditional 2D radiological measures and novel 3D shape descriptors) and signal appearance (extracted from signal intensity histograms) features. The features were validated against manual reference, compared between 2D and 3D MRI scans and used for quantification and classification of IVD degeneration across magnetic resonance datasets containing IVD with early and advanced stages of degeneration. Combination of the novel 3D-based shape and signal intensity features on 3D (area under receiver operating curve (AUC) 0.984) and 2D (AUC 0.988) magnetic resonance data deliver a significant improvement in automated classification of IVD degeneration, compared to the combination of previously used 2D radiological measurement and signal intensity features (AUC 0.976 and 0.983, respectively). Further work is required regarding the usefulness of 2D and 3D shape data in relation to clinical scores of lower back pain. The results reveal the potential of the proposed informatics system for computer-aided IVD diagnosis from MRI in large-scale research studies and as a possible adjunct for clinical diagnosis.

Mesenchymal Stem Cells Mediate Disk Regeneration by Suppression of Fibrosis in Nucleus Pulposus

Introduction Confluent fibrosis, manifested as over-accumulation of fibrous matrix during tissue repair or remodeling, leads to scar formation and often organ failure. Studies suggest that 68% of protruded intervertebral disks and 44% of prolapsed IVD exhibit signs of fibrosis in the nucleus pulposus (NP) and that disk scarring indicates advanced stages of degeneration. It is not clear how fibrosis contributes to the initiation or progression of disk degeneration, and conversely if the prevention of fibrotic events plays a role in IVD repair or regeneration. Mesenchymal stem cells (MSCs) may arrest IVD degeneration and cross-talk with IVD cells via cell-cell contact or long-range signaling, implying MSCs may modulate disk microenvironment and function indirectly. Lupine is a reliable model for IVD degeneration studies as their lumbar spine bears biological relevance to humans regarding mechanics and age-related changes. Studies showed that puncturing to the annulus could transform the NP into a fibrocartilaginous phenotype, recapitulating the fibrotic features of human disk degeneration. We here tested if MSCs can rectify IVD degeneration and its function through modulation of fibrotic events, and investigated the pathways of the action. Materials and Methods Total 6-month-old NZW rabbits with puncture-induced lumbar disk degeneration were intradiscally implanted with Chinchilla bone marrow MSCs, labeled by Qtracker and encapsulated in oligopeptide-hydrogel at 5 x 10E3 cells per disk ( n = 8). Saline or hydrogel alone was injected as control. Treated disk levels were monitored by 3T MRI and radiographs up to 1 year. At 4 to 12 weeks posttreatment, the mechanical integrity of NP and joint segment were investigated by confined compression and motion stiffness tests. The collagen fibril diameter ( n = 120) and the porosity of the fibril meshwork in the NP were analyzed in situ using scanning electron microscopy and morphometric analyzer. The elastic modulus of collagen fibrils ( n = 75) in the NP was measured by nanoindentation using atomic force microscopy. Collagen I and II content was assessed by immunofluorescence. Human bone marrow MSC-conditioned media (MSC-CM) was collected and used to culture alginate-encapsulated NP cells harvested from patients with Pfirrmann grade III/IV disk degeneration. After 7 days, expression of fibrotic genes was studied using real-time PCR ( n = 3). Results T2-weighted MRI and disk height analysis validated that MSCs arrested the degeneration. Motion segment stiffness was significantly attenuated in MSC-treated disks, indicating MSCs alleviated ankylosis caused by the degeneration and restored the spinal segment function. MSCs regenerated the swelling pressure and compressive strength of the NP, implying that MSCs regenerated joint function through preserving the biphasic tissue properties. Collagen molecules in the degenerated NP aggregated into gigantic fibrils, about 2-fold larger in diameter, resulting in fewer fibers and larger interfibrillar space. However, MSCs inhibited the deregulated higher-order assembly of fibrils. MSCs significantly reduced collagen I deposition and prevented the increase of fibril elastic modulus in the NP associated with degeneration. These suggest that the abnormal collagen fibrils are not solely resulted from fibril fusion but also a heterotypic change of collagen composition, and that the positive effects of MSCs are coupled to a regulation of fibrillogenesis in the NP matrix. MSC-CM repressed the expression of fibrosis-related genes MMP12 and HSP47 in degenerative human NP cells. Conclusion MSC transplantation can alleviate fibrotic diseases including pulmonary, renal, and cardiac fibrosis through pathways independent of MSC differentiation. Our findings imply that MSCs also rescue disk degeneration and recover disk function by suppressing fibrotic events. The action of MSCs is elicited through a regulation of fibrillogenesis in part via paracrine signaling to the degenerative NP cells. We propose a model in which MSCs reinforce the mechanical integrity of NP by normalizing the interplay between collagen meshwork and proteoglycan. Disk repair by augmenting the antifibrotic function of MSCs or use of other antifibrotic agents warrants further investigation. This study is supported by the Research Grant Council of Hong Kong and HKU Foundation. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared

Neuronal apoptosis in the striatum of rats treated with 3-nitropropionic acid is not triggered by cell-cycle re-entry

Although terminally differentiated neurons lack the capacity to undergo cell division, they retain the capacity to reactivate the cell cycle. This reactivation, however, has been linked to the degeneration of neurons in many experimental models of neurodegenerative disease and in post-mortem brains of affected patients. Expression of markers of the G1 phase and apoptotic neurons has been detected in the striatal lesion of rats treated with 3-nitropropionic acid (3-NPA). Here we examined whether neuronal apoptosis induced by 3-NPA was mediated by the reactivation of the cell cycle. To this end, we studied whether TUNEL-positive neurons expressed the G1-phase markers cyclin-dependent kinase 4 (CDK4) and cyclin D (CyD). In addition, we also evaluated the neuronal expression of pRb and Ki67 antigens, both of which are involved in the regulation of cell-cycle progression. In 3-NPA-treated rats, CDK4 and CyD were not detected in TUNEL-positive neurons, but they were expressed in neurons in the core of the lesion, which were assumed to be in a more advanced stage of degeneration, since they had weaker NeuN staining and lacked Hoechst staining. In addition, injured neurons in the striatal lesion of 3-NPA-treated rats had lost the constitutive expression of pRb and Ki67 that we had detected in control animals. Taken together, these results indicate that neuronal apoptosis in the striatal lesion of 3-NPA-treated rats was not triggered by cell-cycle re-entry, and we conclude that expression of G1 markers may be considered an aberrant survival response, with no relation to the mechanisms of apoptosis.

Disposal of cell debris from surplus lactotrophs of pituitary gland.

In rats submitted to physiological and experimental stimulation of lactotrophic secretion, as in pregnancy and lactation as well as after estrogen treatment, there is a remarkable proliferation of lactotrophs in the pituitary gland. After interruption of the stimuli, this situation is reversed to basal conditions via a massive programmed cell death of the surplus lactotrophs. The regressive changes occurring in lactotrophs are autolytic in nature, until the advanced stages of degeneration when the cell debris generated by this process are engulfed by phagocytes exhibiting the morphological characteristics of stellate cells. These cells can be seen within the anterior pituitary parenchyma adherent to the wall of capillaries and in the lumen of blood vessels. The number of stellate cells with engulfed lactotroph cell remnants is relatively small in comparison to the massive cell death, an observation that suggests a rapid clearance of cell debris and phagocytic cells from the pituitary parenchyma into the blood stream. The migration of stellate cells from the adenohypophysis involves the degradation of the basement membrane and rearrangement of the capillary wall components. The final disposal of cell debris of regressing lactotrophs within the anterior pituitary therefore appears to be effected by coordination of a pituitary and systemic mononuclear phagocytic system.

Mitochondrial degeneration in dystrophic neurites of senile plaques may lead to extracellular deposition of fine filaments

Recent data show that amyloid precursor protein accumulates inside axons after disruption of fast axonal transport, but how this leads to mature plaques with extracellular amyloid remains unclear. To investigate this issue, primitive plaques in prefrontal cortex of aged rhesus monkeys were reconstructed using serial section electron microscopy. The swollen profiles of dystrophic neurites were found to be diverticula from the main axis of otherwise normal neurites. Microtubules extended from the main neurite axis into the diverticulum to form circular loops or coils, providing a transport pathway for trapping organelles. The quantity and morphology of organelles contained within diverticula suggested a progression of degeneration. Primitive diverticula contained microtubules and normal mitochondria, while larger, presumably older, diverticula contained large numbers of degenerating mitochondria. In advanced stages of degeneration, apparent autophagosomes derived from mitochondria exhibited a loose lamellar to filamentous internal structure. Similar filamentous material and remnants of mitochondria were visible in the extracellular spaces of plaques. This progression of degeneration suggests that extracellular filaments originate inside degenerating mitochondria of neuritic diverticula, which may be a common process in diverse diseases.

Lysosomal cathepsins in embryonic programmed cell death

During limb development, expression of cathepsin D and B genes prefigure the pattern of interdigital apoptosis including the differences between the chick and the webbed digits of the duck. Expression of cathepsin L is associated with advanced stages of degeneration. Analysis of Gremlin− /− and Dkk− /− mouse mutants and local treatments with BMP proteins reveal that the expression of cathepsin B and D genes is regulated by BMP signaling, a pathway responsible for triggering cell death. Further cathepsin D protein is upregulated in the preapoptotic mesenchyme before being released into the cytosol, and overexpression of cathepsin D induces cell death in embryonic tissues by a mechanism including mitochondrial permeabilization and nuclear translocation of AIF. Combined inhibition of cathepsin and caspases suggests a redundancy in the apoptotic molecular machinery, providing evidence for compensatory activation mechanisms in the cathepsin pathway when caspases are blocked. It is concluded that lysosomal enzymes are functionally implicated in embryonic programmed cell death.

Partial urethral obstruction enhances NADPH-diaphorase activity in the monkey (Macaca fascicularis) bladder: light and electron microscopic studies.

The effect of partially obstructing the urethra on the nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) activity in neurons of the intramural ganglia of the monkey (Macaca fascicularis) bladder was examined by light and electron microscopy. Partial urethral ligation was done in adult male monkeys. The animals were sacrificed 2, 4 weeks after partial urethral obstruction. This was compared to controls (normal and sham operated). Urethral obstructed animals were observed to have increased urinary frequency and decreased urinary flow rate. Two weeks after urethral obstruction, the overall NADPH-d activity in the intramural ganglia of the bladder base was enhanced compared to control animals. The frequency of intensely stained NADPH-d positive neurons was increased compared to the control animals. About one-third of intensely stained NADPH-d positive neurons appeared to undergo degenerative changes. At 4 weeks after urethral obstruction, a wide occurrence of NADPH-d positive neurons in advanced stages of degeneration in the bladder base was observed. Cellular debris was strewn among normal looking ganglion cells and along the nerve processes. The proportion of intensely stained NADPH-d positive neurons was relatively lower than the controls. The total number of NADPH-d positive neurons and the nerve fibres in the entire bladder was significantly reduced when compared to control animals. Electron microscopy showed some NADPH-d activity in intramural ganglion cells in 2 weeks after partial urethral obstruction. NADPH-d reaction product (formazan) was deposited on the membranes of the rough endoplasmic reticulum, and the outer membranes of some mitochondria in the intramural neuron. At 4 weeks after urethral obstruction, NADPH-d was present in the membrane of the mitochondria and some mitochondria appeared swollen with disrupted cristae. Present results show that NADPH-d activity in neurons of the intramural ganglia of the monkey (Macaca fascicularis) urinary bladder was increased after two weeks and reduced after 4 weeks of partial urethral obstruction. It is speculated that the increased NADPH-d activity associated with partial urethral obstruction would lead to neuronal damage and death, which may contribute to detrusor overactivity. However, it warrants further investigation to understand the mechanism of neuronal cell death after partial urethral obstruction.

Morphological Changes to Early Stage Taenia solium Cysticerci Following Oxfendazole Treatment

The progressive morphological changes to early stage Taenia solium cysticerci following the treatment of pigs with a single therapeutic dose of oxfendazole (30 mg/kg), are described. On Day 1 after treatment, no obvious changes occurred in the general appearance of the larvae but alternations were seen by electron microscope, with an apparent reduction in the number of microtriches, and a complete disappearance of the tegument. Numerous granules were seen to have accumulated in the tegument cells. As treatment progressed, damage to the cysticerci was more serious and, by five days, all cysticerci were seen to be in an advanced stage of degeneration. By 45 days post-treatment, all cysts were calcified. These results suggest that oxfendazole is a highly effective drug against T. solium cysticerci in the early stages of development.

Circulating autoantibodies recognize and bind dying neurons following injury to the brain.

While it is known that autoimmune cells can protect against cell damage following traumatic injury of the brain, the role of autoantibodies in brain injury is less clear. Here we present evidence in adult rats that following a cortical lesion of the brain, circulating IgG autoantibodies bind to dying neurons in the vicinity of the lesion. At intervals that ranged from 4 h to 7 days after making a unilateral lesion of visual cortex, we observed neurons near the lesion that were immunopositive for rat IgG. Many of these IgG-positive neurons were in advanced stages of degeneration. The magnitude of the immunostaining observed was directly proportional to the percent reactivity to rat IgG of the antibodies that were used. Preadsorption of the antibodies with rat serum eliminated the immunostaining. In addition, immunostaining for serum albumin in sections through the cortical lesion was negative, supporting the conclusion that the positive staining for IgG does not result from the passive diffusion of serum proteins into injured cells. Instead, the evidence presented here strongly suggests that naturally occurring IgG autoantibodies bind specifically to dying neurons in the injured brain. We propose that this autoantibody binding may participate in the phagocytosis and removal of injured neurons.

Development and survival of tyrosine hydroxylase containing neurons in RCS rat retinae

Aim. Dopamine serves a variety of functions in the retina. Abnormalities of the retinal dopaminergic system have been described in the Royal College of Surgeons (RCS) rat as well as other models of retinal degeneration. Dopamine has been implicated in several retinal dysfunctions of retinitis pigmentosa. Dopaminergic amacrine cells respond to light by increasing their tyrosine hydroxylase (TH) activity and the rate of dopamine turnover. This study has, therefore, examined the ontogenesis of TH containing cells in the RCS rat retina to assess whether progressive photoreceptor degeneration affects the development or survival of TH containing cells in any way. Methods. TH immunoreactivity in developing dystrophic RCS rat retinae (postnatal day (PN) 0, 3, 6, 14, 18, 26, 32, 56, 85, 91, 12 month and 15 month) and normal retina (PN day 0, 6, 14, 19, 26, 30, 33, 54 and adults) was compared. Results. TH immunoreactivity in dystrophic retina closely resembled that in normal retina. In both groups, very faintly immunoreactive cells were detected in the proximal retina at PN 0. Immunoreactivity increased until PN 14, when faintly immunoreactive interplexiform (IP) fibers and fibers in the outer plexiform layer could be observed. In both groups, the IP connections reached their mature level of development at about PN 30. Thus the developmental expression of TH immunoreactive cells resembled that of non-dystrophic retina in both chronology as well as types of cells. These cells survived even in the advanced stages of degeneration. Conclusions. The results suggest that the abnormalities in the dopaminergic system of the RCS retinae are not associated with abnormal ontogeny or survival of TH synthesizing cells.