Anders Holtz

Apoptosis and expression of Bcl-2 after compression trauma to rat spinal cord.

We have evaluated by in situ nick-end labeling the presence of apoptotic cells in the spinal cord of rats with compression injury at the level of Th8–9, of mild, moderate, and severe degrees resulting in no neurologic deficit, reversible paraparesis, and paraplegia, respectively. Rats with compression injury surviving 4 or 9 days showed apoptotic glial cells in the longitudinal tracts of the Th8–9, the cranial Th7, and the caudal Th10 segments. The apoptotic cells were most frequently observed in Th7. They did not express glial fibrillar acidic protein (GFAP) and their morphology was compatible with that of oligodendrocytes. Neurons of the gray matter did not present signs of apoptosis. In addition, we studied the immunohisto-chemical expression of Bcl-2, an endogenous inhibitor of apoptosis. Compression induced Bcl-2 immunoreactivity in axons of the long tracts, particularly after moderate and severe compression and 1-day survival. Neurons of dorsal root ganglia were immunoreactive but the neurons of the spinal cord were unstained. The accumulation, presumably caused by arrested axonal transport in sensory pathways, was absent in rats surviving 9 days. In conclusion, compression trauma to rat spinal cord induces signs of apoptosis in glial cells, presumably oligodendrocytes of the long tracts. This may induce delayed myelin degeneration after trauma to the spinal cord. Bcl-2 does not seem to be upregulated in oligodendrocytes

Apoptosis of oligodendrocytes occurs for long distances away from the primary injury after compression trauma to rat spinal cord

Abstract We evaluated by in situ nick end labeling the presence of apoptotic glial cells in the spinal cord of rats which have sustained a moderate and severe compression injury at the level of T8–9, resulting in a severe but reversible paraparesis and irreversible paraplegia, respectively. In a previous investigation we found apoptotic glial cells (oligodendrocytes) in the immediate vicinity of the primary lesion (T7 and T10). The present study was designed to evaluate the extent of such cells in the spinal cord even at long distances away from the primary injury. Rats sustaining a moderate and severe compression injury and surviving 4 and 9 days showed a significant increase in the number of apoptotic glial cells at the T1, T5, T7, T12 and L2 levels. At the T10 level the elevation was significant only after day 9. There was no significant increase in the number of these cells at 4 h and 1 day after moderate and severe compression. In general, the apoptotic cells were most often seen in segments adjacent to the compression. They were randomly located in the ventral, lateral and dorsal tracts but were rarely present in the gray matter of the cord. In conclusion, compression trauma to rat spinal cord induces signs of apoptosis in glial cells, presumably oligodendrocytes of the long tracts. This newly discovered type of secondary injury is widely distributed in the damaged spinal cord and occurs even at long distances remote from the initial compression injury. Apoptotic cell death of oligodendrocytes will induce myelin degeneration and cause additional disturbances of axonal function. This cell damage may be a target for future therapy since it occurs after a delay and chemical compounds are now available by which apoptotic cell death can be modified.

Spinal cord blood flow measured by 14C-iodoantipyrine autoradiography during and after graded spinal cord compression in rats.

The relations between degree of thoracic spinal cord compression causing myelographic block, reversible paraparesis, and extinction of the sensory evoked potential on one hand, and spinal cord blood flow on the other, were investigated. This was done in rats using the blocking weight-technique and 14C-iodoantipyrine autoradiography. A load of 9 g caused myelographic block. Five minutes of compression with that load caused a reduction of spinal cord blood flow to about 25%, but 5 and 60 minutes after the compression spinal cord blood flow was restored to 60% of the pretrauma value. A load of 35 g for 5 minutes caused transient paraparesis. Recovery to about 30% was observed 5 and 60 minutes thereafter. During compression at a load of 55 g, which caused almost total extinction of sensory evoked potential and irreversible paraplegia, spinal cord blood flow under the load ceased. The results indicate that myelographic block occurs at a load which does not cause irreversible paraparesis and that a load which permits sensory evoked potential to be elicited results in potentially salvageable damage.

Spinal cord blood flow changes following systemic hypothermia and spinal cord compression injury : an experimental study in the rat using Laser-Doppler flowmetry

Study design: It is well known that changes of the body temperature as well as trauma influence the blood flow in the brain and spinal cord. However, there is still a lack of knowledge concerning the levels of blood flow changes, especially during hypothermia.Objectives: This investigation was carried out to examine the effects of systemic hypothermia and trauma on spinal cord blood flow (SCBF).Methods: Twenty-four rats were randomized either to thoracic laminectomy only (Th VII–IX) or to 35 g spinal cord compression trauma. The animals were further randomized to either constant normothermia (38°C) or to a systemic cooling procedure, ie reduction of the esophageal temperature from 38 to 30°C. SCBF was recorded 5 mm caudal to the injury zone using Laser-Doppler flowmetry which allows a non-invasive continuous recording of local changes in the blood flow. The autoregulation ability was tested at the end of the experiments by inducing a 30–50 mmHg blood-pressure fall, using blood-withdrawal from the carotid artery.Results: The mean SCBF decreased 2.8% and 3.5% per centigrade reduction of esophageal temperature in the animals sustained to hypothermia with and without trauma, respectively. This could be compared to a decrease of 0.2%/min when only trauma was applied. No significant differences were seen between the groups concerning auto regulatory ability.Conclusions: Our results indicate that the core temperature has a high impact on the SCBF independent of previous trauma recorded by Laser-Doppler flowmetry. This influence exceeds the response mediated by moderate compression trauma alone.Sponsorship: The study was supported by grants from the Laerdal foundation.Spinal Cord (2001) 39, 74–84.

Relation between Spinal Cord Blood Flow and Functional Recovery after Blocking Weight-Induced Spinal Cord Injury in Rats

Spinal cord blood flow (SCBF) and motor performance on the inclined plane were measured up to 9 days after a reversible spinal cord compression injury in 49 Sprague-Dawley rats. A load of 35 g on 11 mm2 of the thoracic spinal cord for 5 minutes caused transient paraparesis with a decrease in the capacity angle on the inclined plane from 62 +/- 1 degree (mean +/- SEM) before injury to 33 +/- 1 degree on Day 1, 45 +/- 2 degrees on Day 4, d and 54 +/- 3 degrees on Day 9. SCBF was measured by the [14C]iodoantipyrine method, and in gray matter there was a decrease from 78.4 +/- 2.3 ml/min/100 g of tissue in uninjured animals to 33.7 +/- 1.5 ml/min/100 g of tissue on Day 1 after injury, increasing to 50.1 +/- 2.0 on Day 4 and to 70.5 +/- 2.7 ml/min/100 g of tissue on Day 9. At the corresponding times, the SCBF values in white matter were 14.5 +/- 0.5, 6.7 +/- 0.5, 10.2 +/- 0.6, and 13.4 +/- 0.6 ml/min/100 g of tissue, respectively. The animals in another group were loaded with 25 g for 5 minutes and on Day 1 exhibited a capacity angle of 43 +/- 2 degrees while the SCBF values for gray and white matter were 55.1 +/- 2.0 and 11.1 +/- 0.4 ml/min/100 g of tissue, respectively; thus, the results in this group were similar to the values on Day 4 in the animals loaded with 35 g.(ABSTRACT TRUNCATED AT 250 WORDS)

Systemic hypothermia following compression injury of rat spinal cord: reduction of plasma protein extravasation demonstrated by immunohistochemistry

Abstract Systemic hypothermia has neuroprotective effects in experimental models of central nervous system ischemia caused by vascular occlusions. The present study addresses the question as to whether systemic hypothermia can influence the extravasation of plasma proteins following severe spinal cord compression trauma using immunohistochemistry to identify the plasma proteins albumin, fibrinogen and fibronectin. Fifteen rats were assigned to one of three groups and received either thoracic (T) laminectomy or severe spinal cord compression trauma of the T8–9 segment. One group comprised laminectomized animals without compression trauma submitted to a hypothermic procedure in which the core temperature was reduced from 38° to 30 °C. The two trauma groups were either submitted to the same hypothermic procedure or kept normothermic during the corresponding time. All animals were killed 24 h following the surgical procedure. The normothermic and hypothermic trauma groups had indications of marked extravasation of albumin, fibrinogen and fibronectin at the site of the injury (T8–9). There was also pronounced extravasation in the cranial and caudal peri-injury zones (T7 and T10) of normothermic injured rats but, with few exceptions, not in the hypothermic ones with the same degree of compression. By measuring the cross-sectional area of the peri-injury zones we found in the hypothermic trauma group a significant reduction of the expansion compared with that present in normothermic injured rats. Our study thus indicates that hypothermia reduces the extravasation of the plasma proteins albumin, fibrinogen and fibronectin following spinal cord compression in the rat. Such a reduction may contribute to neuroprotective effects exerted by hypothermia.

Small-bowel transplantation in the rat with a nonsuture cuff technique: Technical and immunological considerations

Abstract. Small‐bowel transplantation (SBT) using an nonsuture cuff technique was carried out on 137 rats. Preparation of the donor graft was carried out according to conventional procedures. Graft perfusion was done at a fixed pressure of 35 cm water. The left renal vessels of the recipient were dissected, the native kidney removed, and the graft was connected to the vessels by a nonsuture cuff technique. Of the animals, 92% survived for at least 5 days posttransplant. Three different combinations were investigated: (1) isografts; (2) semisyngeneic grafts from nontreated Lewis(LewisDA) Fl hybrids; and (3) semisyngeneic grafts from rabbit antilymphocyte globulin (ALG)‐pretreated Lewis(LewisDA) Fl. In group 1, 80% of the grafts were unaffected after 1 month; flow studies showed slight or no impairment of circulation in the graft. In group 2, the recipients developed clinical signs of graft‐versus‐host disease (GVHD) after 1 week, and at the end of the 2nd week the animals showed signs of severe illness, leading to death due to GVHD. There was also a higher percentage of complications in this group. In group 3, 65% of the animals died. However, 27% showed intact grafts and no signs of GVDH after 1 month, indicating that antibody pretreatment of the donor may successfully prevent GVHD SBT.

Time course of energy perturbation after compression trauma to the spinal cord: An experimental study in the rat using microdialysis

Changes occurring in the extracellular fluid (ECF) concentration of energy-related metabolites were investigated in a well-characterized model of compression trauma to the spinal cord. Microdialysis probes were inserted into exposed grey matter of the dorsal horn at the level of Th 7-8, and perfused with mock cerebrospinal fluid. The trauma was produced 2 hours later by compression of the cord with a 9-, 35-, or 50-g load for 5 min. Microdialysis samples (10-minute fractions) were collected for another 2 hours following decompression. The trauma was associated with an accumulation of lactate, inosine, and hypoxanthine, and an increase in the lactate/pyruvate ratio in the ECF, indicating a profound disturbance in energy metabolism. These changes were related to the severity of spinal cord injury as well as to the spinal cord blood flow (SCBF) reductions and neurological deteriorations previously determined. Following decompression, all ECF metabolites normalized within 20-40 min after mild (9 g) to moderate (35 g) trauma. After severe trauma (50 g), resulting in complete ischemia during compression, followed by irreversible paraplegia, there was a partial recovery of ECF inosine and hypoxanthine, whereas the increase in lactate and the lactate/pyruvate ratio persisted. The results suggest that penumbra conditions prevail during the early posttraumatic period when the degree of trauma results in severe neurological deterioration and that ECF lactate levels in the spinal cord is a sensitive indicator of secondary ischemia after compression injury.

Systemic hypothermia following spinal cord compression injury in the rat: axonal changes studied by β-APP, ubiquitin, and PGP 9.5 immunohistochemistry

Study Design: Systemic hypothermia exerts neuroprotective effects in experimental ischemic CNS models caused by vascular occlusions. Recent experimental and clinical studies have also demonstrated beneficial effects of hypothermic treatment following brain trauma. Objectives: The present study addresses the question as to whether systemic hypothermia has similar protective qualities following severe spinal cord compression trauma using β-APP-, ubiquitin-, and PGP-9.5-immunohistochemistry combined with the ABC complex method as markers to identify axonal changes. Methods: Fifteen rats were randomized into three equally large groups and sustained to either thoracic laminectomy or to severe spinal cord compression trauma of the Th 8–9 segments. The non-trauma group contained laminectomized animals submitted to a hypothermic procedure in which the core temperature was reduced from 38 to 30°C. The two trauma groups were either submitted to the same hypothermic procedure or kept normothermic during the corresponding time. All animals were sacrificed 24 h following the surgical procedure. Results: In the hypothermic non-trauma group no axonal changes were seen. The number of abnormal axons, as indicated by accumulation of immunoreactive material in enlarged axons, was lower in the peri-injury zones of the hypothermic trauma group than in the normothermic trauma group. This difference was most obvious in the cranial peri-injury zones. No differences were seen between the groups in the trauma zones. Conclusions: This study demonstrates reduced axonal swelling in the peri-injury zones of spinal cord injured rats treated with systemic hypothermia. These changes could either indicate neuroprotective effects of the hypothermic treatment, or be results of reduced axonal transport or protein synthesis. To evaluate the clinical importance of our findings, further studies including reliable outcome measures of the animals must be performed.

MAP2 and neurogranin as markers for dendritic lesions in CNS injury. An immunohistochemical study in the rat.

We compared two staining methods for the demonstration of dendrites under normal and pathological conditions of the rat central nervous system. MAP2‐ and neurogranin immunohistochemistry was applied to samples from normal tissue, spinal cord subjected to graded compression trauma, cerebral cortex following contusion trauma, and brains with focal ischemic lesions induced by occlusion of the middle cerebral artery (MCAO). Normal rats showed MAP2 immunoreactivity in nerve cell bodies and dendrites of brain and spinal cord. However, neurogranin staining was present only in nerve cell bodies and dendrites of the normal brain, and not in the spinal cord.