Introduction: This study aimed to analyse immunohis-tochemically the proteolysis of Amyloid Precursor Protein (APP) using Caspase-3-mediated APP proteolytic peptide (CMAP), beta-Amyloid (Aβ) and Active Caspase-3 in post-mortem human specimens in acute and chronic compressive myelopathy.

Compressive myelopathy, occurring through traumatic fracture/dislocation of vertebrae, iatrogenic injury, cervical spondylotic myelopathy (CSM), or metastatic tumour, causes much socio-economic and emotional disability for patients as well as physical consequences. In such conditions, APP is recognised as an early and specifi c marker of axonal injury. The proteolysis of APP in both acute and chronic compressive myelopathy has not yet been described. Studies analysing axonal injury after brain trauma suggest a role for Caspase-3 in the cleavage of APP¹. In addition, Caspase-3-mediated cleavage of APP has been found to be associated with the formation of Aβ, a neurotoxic protein thought to contribute to cell death in Alzheimer’s disease². Furthermore, A? may subsequently encourage activation of Caspases −2, −3, and −6, the major effector molecules in apoptosis². The current study addressed two hypotheses; that APP provides a substrate for the Caspase-3 enzyme, and, that this event is associated with Aβ production in the compressed spinal cord.

Methods: Spinal cord material from 17 patients with documented SCI was analysed. The spatial distribution of cellular immunoreactivity was qualitatively assessed in injury due to trauma (n=5), iatrogenic event (n=1), CSM (n=6) and metastatic tumour (n=5). Morphological, immunohistochemical and immunofl uorescent techniques were used to investigate APP proteolysis.

Results: Caspase-3, APP, CMAP and Aβ were present in anterior horn cells of the grey matter and axons of the white matter. An association was found between neuronal immunoreactivity and that of axons in motor tracts. Dual-immunolabelling revealed axonal co-localisation of CMAP with Aβ and Caspase-3 with Aβ. Although CMAP was present in axons which were immunoposi-tive for APP, an inverse relationship was found as each marker was limited to its own, distinct region, consistent with the theory that CMAP actively cleaves APP. In neurons, co-localisation occurred between Caspase-3 and Aβ, and CMAP with Aβ. No neuronal co-localisation was shown between CMAP and APP in the acute and chronic state.

Discussion: Caspase-3 appears likely to contribute to the proteolytic cleavage of APP in compressive myelop-athy. CMAP was associated with the production of Aβ as demonstrated using single and dual immunolabelling. Furthermore, evidence is given for the association of Caspase-3 itself with the neurotoxic peptide, Aβ. It is possible that activation of Caspase-3 via these secondary mechanisms may trigger the advancement of the apoptotic cascade with the subsequent demise of the cell.

Introduction: Apoptosis, or secondary cell death, has been demonstrated in a number of neurological conditions, including Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis and brain ischaemia. It is well established from studies of acute spinal cord injury that apoptosis seems an important factor in secondary cell death and irreversible neurological deficit. It is only recently that studies have emerged analysing secondary cell death in chronic injury to the cord. In this study, the spatial and temporal expression of apoptotic cells was analysed in acute traumatic spinal cord injury (SCI) (n=6) and chronic myelopathies due to metastatic tumour (n=5), degenerative spondylosis (n=6) and syringomyelia (n=4). The study aimed to demonstrate apoptosis in compressive spinal cord injury and to analyse the spatial and temporal distribution of apoptosis in acute and chronic myelopathy.

Method: Archival material from 21 spinal cords of patients with documented myelopathy during life and definitive evidence on post mortem examination were available for study. The spatial and temporal expression of apoptotic cells was analysed in acute traumatic spinal cord injury (SCI) (n=6) and chronic myelopathy due to metastatic tumour (n=5), degenerative spondylosis (n=6) and syringomyelia (n=4).

Immunohistochemical analysis of each specimen was conducted using markers of apoptosis, as well as the biochemical apoptotic marker TUNEL. A total of 1800 histopathological slides were analysed. Specimens were also analysed using confocal microscopy to identify the immunopositive cell type. A combination of morphological, immunohistochemical and in situ end-labelling techniques were used to investigate the mechanism of cell death in this experiment. The analytical techniques employed were aimed at showing firstly the presence of apoptosis and secondly the size and position of the damaged regions.

Results: Positivity for active Caspase-3, DNA-PKCS, PARP, TUNEL and active Caspase-9 was found in glia (oligodendrocytes and microglia) axons and neurons in both acute and chronic compression above, below and at the site of compression. In chronic compression, the severity of positivity for apoptotic immunological markers was positively correlated with the severity of white matter damage, as measured by APP immunostaining for axonal injury, and Wallerian degeneration. There was no correlation between the duration of chronic compression and immunopositivity for apoptotic markers. In acute SCI, axonal swellings were consistently positive for Caspases −9 and -3, suggesting mitochondrial activation of apoptotic pathways.

Conclusion: Apoptosis occurs in both acute and chronic spinal cord injury. In acute compression, axonal injury is associated with apoptotic immunopositivity of glia and neurons. In chronic compression, apoptosis of oligodendrocytes and microglia correlates with demyelination of axons within the white matter.

Introduction: Although it is well recognized that the outer annulus is innervated, the relative densities of innervation of different regions of the disc have not been quantitated. We present here the first comparative analysis of the innervation of the innervation of different regions of the lumbar intervertebral disc.

Methods: A sheep model was used allowing evaluation of the whole motion segment. Four sheep spines were used. One was processed for PGP 9.5 immunoflourescence and three were processed for PGP 9.5 immunoperoxidase histochemistry. Serial sagittal sections were obtained and a count was made of the densities of innervation of different regions of the endplate and annulus. These were compared to identify which areas of the disc and endplate are most innervated.

Results: The endplate innervation is concentrated centrally adjoining the nucleus. The mean density of innervation of the central endplate was 0.44 (SEM 0.07) nerves/ mm² while the mean density of the peripheral endplate was 0.10 (SEM 0.03) nerves/ mm² (p= 0.0001). There was no significant difference between the overall endplate and annulus innervation densities 0.52 (SEM 0.1) v 0.37 (SEM 0.07) p=0.2. But the peri-annular connective tissue, external to the outer annulus contained the densest innervation of any region in the motion segment 1.05 (SEM 0.16).

Discussion: The lumbar intervertebral disc has a meagre innervation. This is concentrated in the peri-annular connective tissue and the central endplate. While receptor threshold is more closely related to nociceptive function than innervation density, these findings have important implications for any treatment of discogenic pain.

INTRODUCTION: Apoptosis, or secondary cell death, has been demonstrated in a number of neurological conditions, including Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis and brain ischaemia. It is well established from studies of acute spinal cord injury that apoptosis seems an important factor in secondary cell death and irreversible neurological deficit. It is only recently that studies have emerged analysing secondary cell death in chronic injury to the cord. In this study, the spatial and temporal expression of apoptotic cells was analysed in acute traumatic spinal cord injury (SCI) (n=6) and chronic myelopathies due to meta-static tumour (n=5), degenerative spondylosis (n=6) and syringomyelia (n=4). The study aimed to demonstrate apoptosis in compressive spinal cord injury and to analyse the spatial and temporal distribution of apoptosis in acute and chronic myelopathy.

METHOD: Archival material from 21 spinal cords of patients with documented myelopathy during life and definitive evidence on post mortem examination were available for study. The spatial and temporal expression of apoptotic cells was analysed in acute traumatic spinal cord injury (SCI) (n=6) and chronic myelopathy due to metastatic tumour (n=5), degenerative spondylosis (n=6) and syringomyelia (n=4).

Immunohistochemical analysis of each specimen was conducted using markers of apoptosis, as well as the biochemical apoptotic marker TUNEL. A total of 1800 histopathological slides were analysed. Specimens were also analysed using confocal microscopy to identify the immunopositive cell type. A combination of morphological, immunohistochemical and in situ end-labelling techniques were used to investigate the mechanism of cell death in this experiment. The analytical techniques employed were aimed at showing firstly the presence of apoptosis and secondly the size and position of the damaged regions.

RESULTS: Positivity for active Caspase-3, DNA-PKCS, PARP, TUNEL and active Caspase-9 was found in glia (oligodendrocytes and microglia) axons and neurons in both acute and chronic compression above, below and at the site of compression. In chronic compression, the severity of positivity for apoptotic immunological markers was positively correlated with the severity of white matter damage, as measured by APP immunostaining for axonal injury, and Wallerian degeneration. There was no correlation between the duration of chronic compression and immunopositivity for apoptotic markers. In acute SCI, axonal swellings were consistently positive for Caspases −9 and -3, suggesting mitochon-drial activation of apoptotic pathways.

CONCLUSION: Apoptosis occurs in both acute and chronic spinal cord injury. In acute compression, axonal injury is associated with apoptotic immunopositivity of glia and neurons. In chronic compression, apoptosis of oligodendrocytes and microglia correlates with demyelination of axons within the white matter.

INTRODUCTION: Although it is well recognised that the outer annulus is innervated, the relative densities of innervation of different regions of the disc have not been quantitated. We present here the first comparative analysis of the innervation of the innervation of different regions of the lumbar intervertebral disc.

METHODS: A sheep model was used allowing evaluation of the whole motion segment. Four sheep spines were used. One was processed for PGP 9.5 immunofluorescence and three were processed for PGP 9.5 immunoperoxidase histochemistry. Serial sagittal sections were obtained and a count was made of the densities of innervation of different regions of the endplate and annulus. These were compared to identify which areas of the disc and endplate are most innervated.

RESULTS: The endplate innervation is concentrated centrally adjoining the nucleus. The mean density of innervation of the central endplate was 0.44 (SEM 0.07) nerves/mm2 while the mean density of the peripheral endplate was 0.10 (SEM 0.03) nerves/ mm² (p= 0.0001). There was no significant difference between the overall endplate and annulus innervation densities 0.52 (SEM 0.1) v 0.37 (SEM 0.07) p=0.2. But the peri-annular connective tissue, external to the outer annulus contained the densest innervation of any region in the motion segment 1.05 (SEM 0.16).

DISCUSSION: The lumbar intervertebral disc has a meagre innervation. This is concentrated in the peri-annular connective tissue and the central endplate. While receptor threshold is more closely related to noci-ceptive function than innervation density, these findings have important implications for any treatment of discogenic pain.