Relationship between intrathecal oxygen tension and ultrastructural changes in the spinal cord during experimental aortic clamping.

OBJECTIVES: To investigate spinal cord ultrastructure related to cerebrospinal fluid (CSF) oxygenation. DESIGN: experimental aortic occlusion model with intrathecal oxygen tension monitoring. MATERIALS AND METHODS: Two groups of pigs underwent proximal (P) or double (D) aortic occlusion for 30 min followed by 1 h of reperfusion. In a third group (I) segmental arteries distal to T3 were clamped for 90 min. A thin pO(2), pCO(2) and pH sensor was placed intrathecally for continuous monitoring of CSF. Spinal cord segments were studied by electron microscopy (EM). RESULTS: In group P, CSF-pO(2)rapidly decreased during clamping and major changes in pH and pCO(2)were seen. EM demonstrated neuronal degeneration with loss of cellular integrity and severe affection of organelles. In the group D, CSF oxygenation decreased to about half, but with only moderate changes in the metabolic parameters. Group I showed no significant changes in CSF measurements. The latter groups were similar at EM, showing only mild mitochondrial changes. CONCLUSIONS: The level of CSF oxygenation during aortic cross-clamping or segmental artery interruption seems to correlate with ultrastructural changes in the spinal cord. This online intrathecal monitoring technique may provide valuable information on spinal cord circulation during thoracoabdominal aortic surgery.

Spinal cord morphology after chronic intrathecal administration of adenosine in the rat.

BACKGROUND: The endogenous compound adenosine is known to have various modulatory effects both in the peripheral and central nervous system. Adenosine and its analogues induce antinociception in animal models when administrated systemically and intrathecally (IT), both in acute and chronic models of pain. Before a new drug is introduced for spinal pain treatment in humans, experimental studies of neurotoxicity must be undertaken. METHODS: This study was performed in rats in order to reveal morphological or morphometric signs of spinal cord damage after chronic (two weeks) administration of adenosine. After insertion of IT catheters, the animals were injected twice a day during two weeks with adenosine (100 microg) or saline (controls). Potential spinal neurotoxicity was evaluated morphologically by light and electron microscopy supplemented by a morphometric analysis. RESULTS: There were no signs of histologic changes indicating neurotoxic effects by any of the methods of analysis. The morphological findings in the adenosine treated rats did not differ in any case from those of the saline treated animals. CONCLUSION: The results suggest that chronic IT administration of a high dose of adenosine is not associated with neurotoxicity in the rat spinal cord.

Sameridine--intrathecal injection in the rat. Morphometric and morphologic analysis after chronic administration and effects on spinal cord blood flow.

BACKGROUND: Sameridine is a type of compound with both local anaesthetic and analgesic effects with the clinical intention to be used intrathecally (i.t.) in order to provide both surgical anaesthesia and prolonged postoperative analgesia. Before new drugs are introduced for clinical use, they must be tested for potential toxic effects. METHODS: In the present study sameridine (5 or 10 mg/ml), bupivacaine (5 mg/ml) or saline (9 mg/ml) was injected intrathecally in rats twice, daily (at 07:00 and 19:00), 5 days a week for 2 weeks. Thereafter, the rats were anaesthetised, perfused and the spinal cords were prepared for microscopic investigation. A morphologic method, using light and electron microscopic examination of the cross-section of the spinal cord, was combined with a quantitative morphometric analysis of the number and size of neuronal cells in the dorsal horn as a sensitive indicator of neurotoxicity. Using the laser-Doppler flowmetry technique, the effects of saline and sameridine (1, 5 and 10 mg/ml) on spinal cord blood flow (SCBF) was studied. RESULTS: No signs of neurotoxicity could be seen in any of the animals and no significant differences were seen when comparing the cell number or cell sizes in the groups injected with sameridine, bupivacaine or saline. After i.t. administration of 10 mg/ml sameridine a significant, short-lasting, decrease in SCBF (72% of pre-drug value) was seen. CONCLUSION: In conclusion, our studies do not show any signs of neurotoxic effects of i.t. administration of sameridine in the rat. A transient decrease in SCBF was noted after i.t. injection of sameridine 10 mg/ml.

Ventilation with constant versus decelerating inspiratory flow in experimentally induced acute respiratory failure.

BACKGROUND: Recognition of the potential for ventilator-associated lung injury has renewed the debate on the importance of the inspiratory flow pattern. The aim of this study was to determine whether a ventilatory pattern with decelerating inspiratory flow, with the major part of the tidal volume delivered early, would increase functional residual capacity at unchanged (or even reduced) inspiratory airway pressures and improve gas exchange at different positive end-expiratory pressure levels. METHODS: Surfactant depletion was induced by repeated bronchoalveolar lavage in 13 anesthetized piglets. Decelerating and constant inspiratory flow ventilation was applied at positive end-expiratory pressure levels of 22, 17, 13, 9, and 4 cm H(2)O. Tidal volume, inspiration-to-expiration ratio, and ventilatory frequency were kept constant. Airway pressures, gas exchange, functional residual capacity (using a wash-in/washout method with sulfurhexafluoride), central hemodynamics, and extravascular lung water (using the thermo-dye-indicator dilution technique) were measured. RESULTS: Decelerating inspiratory flow yielded a lower arterial carbon dioxide tension compared to constant flow, that is, it improved alveolar ventilation. There were no differences between the flow patterns regarding end-inspiratory occlusion airway pressure, end-inspiratory lung volume, static compliance, or arterial oxygen tension. No differences were seen in hemodynamics and oxygen delivery. CONCLUSIONS: The decelerating inspiratory flow pattern increased carbon dioxide elimination, without any reduction of inspiratory airway pressure or apparent improvement in arterial oxygen tension. It remains to be established whether these differences are sufficiently pronounced to justify therapeutic consideration.

Chronic subarachnoid midazolam (Dormicum) in the rat. Morphologic evidence of spinal cord neurotoxicity.

BACKGROUND AND OBJECTIVES: In humans, the benzodiazepine midazolam has been reported to exert an antinociceptive action after subarachnoid injections. It has been shown that subarachnoid midazolam given to rabbits produces significant pathology in spinal cord morphology, as detected with light microscopy. In order to further characterize these changes, this study was performed, using a more sensitive histologic technique, including electron microscopy as well as unbiased morphometry. METHODS: The histopathology of the rat lumbar spinal cord was investigated after chronic subarachnoid administration of a commercially available preparation of midazolam. After daily injections of 100 micrograms of midazolam, the animals were transcardially perfused on the twentieth day with a mixture of formaldehyde and glutaraldehyde. RESULTS: Morphometric evaluation of cell number and mean cell volume (MCV) by the disector method revealed a significant lower (P < .05) cell number and a tendency toward higher MCV in the midazolam-injected group (n = 6), compared to the rats injected with saline (n = 6). The higher MCV, in combination with a reduced number of nerve cells, indicated a loss of small neurons. The electron microscopic findings confirmed that midazolam caused neuronal death, since degenerated cell somata, fibers, and terminals were observed in most of the rats. Furthermore, an increased number of microglial cells phagocytosing nerve structures were also seen mainly in the dorsal horn. CONCLUSIONS: The authors found that chronic subarachnoid administration of midazolam gives objective signs of neurotoxicity in the rat spinal cord. The authors' findings are in contrast to those of an earlier light microscopic study in the rat. The present results emphasize both the necessity of morphometric and ultrastructural studies before spinal administration of novel drugs to humans and the neurotoxic potential of midazolam. Since neurotoxicity of midazolam now has been demonstrated in both rats and rabbits, there may be reason to be sceptical of the use of subarachnoid midazolam in humans.

Different ventilatory approaches to keep the lung open.

OBJECTIVES: To study the ability of different ventilatory approaches to keep the lung open. DESIGN: Different ventilatory patterns were applied in surfactant deficient lungs with PEEP set to achieve pre-lavage PaO2. SETTING: Experimental laboratory of a University Department of Anaesthesiology and Intensive Care. ANIMALS: 15 anaesthetised piglets. INTERVENTIONS: One volume-controlled mode (L-IPPV201:1.5) and two pressure-controlled modes at 20 breaths per minute (bpm) and I:E ratios of 2:1 and 1.5:1 (L-PRVC202:1 and L-PRVC201.5:1), and two pressure-controlled modes at 60 bpm and I:E of 1:1 and 1:1.5 (L-PRVC601:1 and L-PRVC601:1.5) were investigated. The pressure-controlled modes were applied using "Pressure-Regulated Volume-Controlled Ventilation" (PRVC). MEASUREMENTS AND RESULTS: Gas exchange, airway pressures, hemodynamics, FRC and intrathoracic fluid volumes were measured. Gas exchange was the same for all modes. FRC was 30% higher with all post-lavage settings. By reducing inspiratory time MPAW decreased from 25 cmH2O by 3 cmH2O with L-PRVC201.5:1 and L-PRVC601:1.5. End-inspiratory airway pressure was 29 cmH2O with L-PRVC201.5:1 and 40 cmH2O with L-IPPV201:1.5, while the other modes displayed intermediate values. End-inspiratory lung volume was 65 ml/kg with L-IPPV201:1.5, but it was reduced to 50 and 49 ml/kg with L-PRVC601:1 and L-PRVC601:1.5. Compliance was 16 and 18 ml/cmH2O with L-PRVC202:1 and L-PRVC201.5:1, while it was lower with L-IPPV201:1.5, L-PRVC601:1 and L-PRVC601:1.5. Oxygen delivery was maintained at pre-lavage level with L-PRVC201.5:1 (657 ml/min.m2), the other modes displayed reduced oxygen delivery compared with pre-lavage. CONCLUSION: Neither the rapid frequency modes nor the low frequency volume-controlled mode kept the surfactant deficient lungs open. Pressure-controlled inverse ratio ventilation (20 bpm) kept the lungs open at reduced end-inspiratory airway pressures and hence reduced risk of barotrauma. Reducing I:E ratio in this latter modality from 2:1 to 1.5:1 further improved oxygen delivery.

Histopathology after repeated intrathecal injections of preservative-free ketamine in the rabbit: a light and electron microscopic examination.

Epidural and spinal administration of ketamine has been used in humans. Single-dose studies have shown that preservative-free ketamine lacks neurotoxic effects, but there are no studies after repeated administrations. The aim of this study was to examine the effects of daily administration of preservative-free ketamine. Fourteen New Zealand albino rabbits were assigned to two groups receiving either intrathecal preservative-free ketamine 5 mg, 0.5 mL 1% solution (eight rabbits) or saline 0.5 mL (six rabbits) once a day for 14 consecutive days. The rabbits had a total subcutaneous implanted intrathecal catheter, which was introduced during general anesthesia. On Day 15 the rabbits were anesthetized and in vivo fixated by transcardial perfusion with Tyrode's solution followed by a mixture of 2% glutaraldehyde and 1% formaldehyde in a 0.1 mol/L phosphate buffer. A segment 5 cm on each side of the catheter tip was removed and kept in a cold solution of the fixative. Light microscopic, electron microscopic, and morphometric examinations showed no differences between the spinal cords from the rabbits injected with ketamine versus saline. Intrathecal ketamine produced motor impairment for a period of 15 min. We conclude that repeated intrathecal administration of preservative-free ketamine confirms the lack of neurotoxicity from single-dose studies.

Intrathecal injection of lysine acetylsalicylic acid in the rat: a neurotoxicological study.

Lysine acetylsalicylic acid has been reported to induce analgesic effects in humans after intrathecal (i.t.) injection. Before conducting further studies in humans with this drug, it is important to evaluate potential toxicological effects on the spinal cord in animals. In the present study the effects of chronic intrathecal administration of provocative doses of lysine acetylsalicylic acid (L-ASA) on the rat spinal cord were evaluated using light and electron microscopy and a quantitative morphometric method. We also investigated the effects of single doses of the drug on the spinal cord blood flow (SCBF) using the laser-Doppler flowmetry technique. No histopathological changes or differences in number or density of neuronal cells could be seen after chronic administration of L-ASA as compared to controls. The SCBF decreased immediately after i.t. injection of a large dose (4 mg) of L-ASA and returned to predrug levels within 10 min. At the end of the experiment metabolic acidosis was detected, indicating a systemic effect of acetylsalicylic acid. It is concluded that no neurotoxic effects on the spinal cord were seen after chronic i.t. injection of L-ASA. From a neurotoxicological point of view, our findings do not contraindicate the spinal use of L-ASA in humans.

A neurotoxicologic evaluation of the spinal cord after chronic intrathecal injection of R-phenylisopropyl adenosine (R-PIA) in the rat.

Studies in animals have shown that the adenosine receptor agonist R-phenylisopropyl adenosine (R-PIA) induces antinociceptive effects after intrathecal administration. Before such a potentially antinociceptive drug could be considered for intrathecal injection in humans, a neurotoxicologic examination of the spinal cord must be performed in animal models. Rats were injected once every day for 14 consecutive days with R-PIA or saline (controls). The number and density of neuronal cells were calculated by using light microscopy, and further examined with electron microscopy. The "disector method," which is an unbiased stereologic estimator of cell number and mean cell volume, was used for quantitative morphometric analyses. With this technique no significant changes could be seen in rats that had received R-PIA as compared to control rats. We conclude that no significant histologic changes could be detected after chronic intrathecal administration of R-PIA.

Repeated intrathecal injections of dezocine produce antinociception without evidence for neurotoxicity in the rat: a study of morphometric evaluation of spinal cord histology.

This study was designed to determine the antinociceptive and spinal cord histologic effects of a new agonist/antagonist opioid drug dezocine. This drug was injected intrathecally in rats at a dose of 50 or 125 micrograms twice daily for 14 days. The tail-flick test showed that the antinociceptive effect declined gradually, with no detectable effects by day 14. Quantitative histologic techniques and light and electron microscopy showed that neither dose, compared with vehicle, created any morphologic changes in the spinal cord that could be attributed to a neurotoxic or otherwise degenerative effect of the drug. In conclusion, dezocine is a drug that gives rise to sustained antinociceptive effects when administered intrathecally and causes no morphologic changes in the rat spinal cord that could be indicative of neurotoxic potential.

Histopathology and evaluation of potentiation of morphine-induced antinociception by intrathecal droperidol in the rat.

Clinical studies have indicated that a low dose of the dopamine D2-receptor antagonist droperidol given epidurally potentiate the antinociceptive effect of epidural morphine. The present study was conducted in order to evaluate this drug interaction in a rat model. Rats were given morphine and droperidol intrathecally in several combinations of doses. It was found that droperidol had no antinociceptive effect by itself, nor in combination with morphine. It was also shown that droperidol and morphine exert no histopathological effects on the rat spinal cord. This discrepancy between clinical findings and experimental pain studies suggests different modes of action of droperidol in the two situations.

Studies on the development of tolerance and potential spinal neurotoxicity after chronic intrathecal carbachol-antinociception in the rat.

The antinociceptive effect in rats produced by chronic intrathecal administration of carbachol was studied for 14 days by the tail immersion test and the paw compression test. Daily injection of 10 micrograms carbachol intrathecally (lumbar level) to 8 rats produced an increase in latency times lasting from day 1 to day 4. The effect was statistically significant during the first 4 days, but not thereafter in both the tail immersion test and the paw compression test as compared to the rats (n = 6) injected with saline. Histopathological examinations of the lumbar spinal cord by light and electron microscopy revealed no signs of neurotoxic reactions of the neurons, nor the spinal tracts. Quantitative morphometric analyses were made by the "disector method", which is an unbiased stereological estimator of cell number and mean cell volume. In the laminae I-III of the L:1 segment, an average number of 88,000 cells/mm3 was found and the mean cell volume was calculated at 560 microns3. Comparison with untreated rats (n = 4) and those injected with saline showed no statistically significant differences. In the present study, the combination of different morphological analyses offers a sensitive method to trace toxic reactions of the nervous tissue. According to these results, intrathecal carbachol produces antinociception, and seems atoxic to spinal nervous tissue, but before intrathecal administration of carbachol to humans is considered, more neurotoxicological data must be obtained.

An experimental study of different ventilatory modes in piglets in severe respiratory distress induced by surfactant depletion.

In 19 anesthetized piglets 3 ventilatory modes were studied after inducing pulmonary insufficiency by bronchoalveolar lavage by the method of Lachmann. The lavage model was considered suitable for reproduction of severe respiratory distress. This model was reproducible and stable with respect to alveolar collapse, decrease in static chest-lung compliance and increase in extravascular lung water. The ventilatory modes studied were volume-controlled intermittent positive-pressure ventilation (IPPV), pressure-controlled inverse ratio ventilation (IRV), and pressure-controlled high-frequency positive-pressure ventilation (HFPPV). The 3 ventilatory modes were used in random sequence for at least 30 min to produce a ventilatory steady state. Ventilation with no PEEP, permitting alveolar collapse, was interposed between each experimental mode. The ability to open collapsed alveoli, i.e. alveolar recruitment, was different. The recruitment rate for IPPV was 74%, but for IRV and HFPPV it was 95%, respectively. Although IRV provided the best PaO2, this was at the expense of high airway pressures with circulatory interference and reduced oxygen transport. In contrast to this, HFPPV provided lower airway pressures, less circulatory interference and improved oxygen transport. In the clinical setting there might be negative effects on vital organs and functions unless the ventilatory modes are continuously and cautiously adapted to the individual requirements in different phases of severe respiratory distress. Therefore, one ventilatory strategy could be to "open the airways" with IRV, but then switch to HFPPV in an attempt to maintain the airways open with lesser risk of barotrauma and with improved oxygen transport.

Quantitative ultrastructural study of boutons of ascending afferents to the feline lateral cervical nucleus.

A quantitative ultrastructural study has been performed of 300 labelled and the same number of unlabelled boutons in the feline lateral cervical nucleus (LCN) of 5 adult cats after cervical or lumbar injections of lectin-conjugated horseradish peroxidase and histochemical reaction with tetramethylbenzidine. On an average the labelled boutons were slightly elongated, had areas of 2.25 micron 2, mitochondrial volume fractions of 11.2% and densities of synaptic vesicles of 35.4.micron-2. The synaptic vesicles were round to flattened with a mean length of 60 nm and length/width ratios of 1.21. The labelled boutons were significantly larger and had significantly lower densities of synaptic vesicles than the unlabelled boutons. The labelled boutons constituted about 14% of the bouton volume of the investigated areas of the LCN. Most of them were axo-dendritic and about 16% synapsed with cell bodies with ultrastructural characteristics similar to the cervico-thalamic projection neurones of the LCN. The number of boutons labelled after the injections comprising 3-5 adjacent segments of the spinal cord was calculated to 6.8 X 10(6). Based on the assumption that they represented terminals of spinocervical tract cells, it was calculated that each of these cells in the lumbar cord gives rise to an average of 4400 boutons in the LCN.

Ultrastructure of spinal efferents to the lateral reticular nucleus: an EM study using anterograde transport of WGA-HRP complex.

Anterograde transport of lectin-conjugated horseradish peroxidase and subsequent incubation with tetramethylbenzidine were employed to label the spinal terminals within the feline lateral reticular nucleus (NRL) for ultrastructural identification. Quantitative studies demonstrated that compared to the unlabelled terminals the spinal boutons were more than twice as large and contained fewer synaptic vesicles. Most of the synaptic contacts of the labelled terminals were located on dendritic shafts but contacts on dendritic spines as well as perikarya were also present. In four cases (all with cervical injections), the postsynaptic cells could be studied in the transversal sections of the medulla in the nuclear plane. The neurons were large and elongated with longest and shortest diameters of about 60 X 30 microns, belonging to the largest category of cells within the NRL. The observations were discussed and related to the findings made in other studies of the NRL.

Fluorescent double-labelling study of ascending and descending neurones in the feline lateral cervical nucleus.

The organization of ascending and descending neurones of the lateral cervical nucleus (LCN) was investigated in 10 adult cats after injections of the fluorescent tracers Fast Blue and Nuclear Yellow. Injections into the thalamus and tectum resulted in up to 3000 labelled cell profiles within the contralateral LCN. This corresponded to a calculated number of 4500 labelled LCN neurones. The greatest diameter of the labelled cell profiles was about 30 micron. They were located throughout the nucleus, but were less numerous in its medial portion. Injections mainly into the dorsal horn of different pairs of cervical and lumbar segments of the spinal cord resulted in a calculated number of up to 305 labelled LCN cells. The diameter of these cell profiles was about 25 micron and they were mainly situated in the rostro-ventral and medial parts of the LCN. Double-labelled cells with ascending and descending projections were not encountered after injections into the thalamus-tectum and spinal segments C5-6. About 15% of the descending LCN cells were double-labelled by pairs of spinal injections separated by intervals of one segment. It is concluded that the neurones descending down the spinal cord and ascending to the thalamus-tectum constitute different subpopulations of cells within the LCN and that a minor proportion of the descending cells seem to project to at least three adjacent segments of the spinal cord.

Light and electron microscopic study of neurones in the feline lateral cervical nucleus with a descending projection.

Nerve cells in the feline lateral cervical nucleus (LCN) with a descending projection were labelled by injections of horseradish peroxidase, in most cases conjugated to wheat germ agglutinin. After small injections into different spinal cord segments in 16 cats the labelled cells were found mainly in the rostral and ventral portions of the ipsilateral LCN, without a detectable topographic organization. Massive bilateral injections were made into the cervical or lumbar enlargement in 6 cats. Unilateral lesions of the dorsolateral funiculus rostral to the injections generally prevented labelling of LCN cells on the same side. The other side was used to calculate the total number of descending LCN neurones and it was estimated that approximately 500 cells projected down the spinal cord. Comparison with the number of labelled cells after the small injections revealed that the descending LCN neurones projected to an average of at least two spinal segments. Quantitative ultrastructural analyses were made of 17 descending neurones from 7 cats subjected to massive unilateral injections into the cervical or lumbar enlargement. The labelled cells constituted a fairly homogeneous population with respect to the investigated somatic and dendritic features. The morphology and relative frequency of the descending neurones indicate that they may constitute parts of the subpopulation of small LCN cells that have previously been assumed to consist of locally ramifying interneurones.

Somatotopic termination of spinal afferents to the feline lateral cervical nucleus.

The labelling pattern of the feline lateral cervical nucleus (LCN) was investigated after pressure injections of lectin-conjugated horseradish peroxidase into cervical, thoracic or lumbar segments of the spinal cord. Sixteen cats received a 5-8 nl injection staining large parts of mainly the ipsilateral grey matter of a single segment. Light microscopic examination of frozen sections reacted with tetramethylbenzidine showed a somatotopic organization in the LCN. Rostral segments of the spinal cord projected mainly to rostroventral and medial parts of the ipsilateral LCN, while more caudally located segments projected to more dorsocaudal and lateral parts of the nucleus. Minor contralateral labelling with a similar somatotopic arrangement was seen in animals given cervical and lumbar injections. No significant labelling was found in the LCN of three control animals, the segmental injections of which were engaged mainly into the ipsilateral dorsal columns and the dorsolateral funiculus. Ultrastructural analysis in two animals which received multiple cervical or lumbar injections showed that about 70% of the peroxidase-positive structures in the LCN were boutons and the rest small myelinated axons. The precise termination pattern of ascending afferents to the LCN is compatible with the somatotopic organization of the other relay centres in the spino-cervico-thalamic pathway.

Endogenous peroxidase-like activity in the feline dorsal column nuclei and spinal cord.

Endogenous peroxidase-like activity was investigated with a combined light and electron microscopical technique in 15 cats. The lateral cervical nucleus, the dorsal column nuclei, and segments C6 and L5 of the spinal cord were incubated with diaminobenzidine-tetrahydrochloride (DAB) or tetramethylbenzidine (TMB). After histochemical reaction with DAB a considerable amount of activity was found in nerve cells, astrocytes and pericytes. The neuronal labelling was mainly located in mitochondria of axon terminals and in dendrites whereas the astrocytic and pericytic activity was found in cytoplasmic dense bodies. The quantity of stained structures differed considerably between the animals. In TMB reacted tissue endogenous peroxidase-like activity was only sparsely seen. It was found mainly in frozen sections, in which the neuropil and perivascular structures sometimes contained granules and irregular filaments. The significance of the findings is discussed in relation to observations in tracer studies using horseradish peroxidase.