Fajko F. Bajrović

Interactions between cardiac, respiratory and EEG-δ oscillations in rats during anaesthesia

We hypothesized that, associated with the state of anaesthesia, characteristic changes exist in both cardio‐respiratory and cerebral oscillator parameters and couplings, perhaps varying with depth of anaesthesia. Electrocardiograms (ECGs), respiration and electroencephalograms (EEGs) were recorded from two groups of 10 rats during the entire course of anaesthesia following the administration of a single bolus of ketamine–xylazine (KX group) or pentobarbital (PB group). The phase dynamics approach was then used to extract the instantaneous frequencies of heart beat, respiration and slow δ‐waves (within 0.5–3.5 Hz). The amplitudes of δ‐ and θ‐waves were analysed by use of a time–frequency representation of the EEG signal within 0.5–7.5 Hz obtained by wavelet transformation, using the Morlet mother wavelet. For the KX group, where slow δ‐waves constituted the dominant spectral component, the Hilbert transform was applied to obtain the instantaneous δ‐frequency. The θ‐activity was spread over too wide a spectral range for its phase to be meaningfully defined. For both agents, we observed two distinct phases of anaesthesia, with a marked increase in θ‐wave activity occurring on passage from a deeper phase of anaesthesia to a shallower one. In other respects, the effects of the two anaesthetics were very different. For KX anaesthesia, the two phases were separated by a marked change in all three instantaneous frequencies: stable, deep, anaesthesia with small frequency variability was followed by a sharp transition to shallow anaesthesia with large frequency variability, lasting until the animal awoke. The transition occurred 16–76 min after injection of the anaesthetic, with simultaneous reduction in the δ‐wave amplitude. For PB anaesthesia, the two epochs were separated by the return of a positive response to the pinch test at 53–94 min, following which it took a further period of 45–70 min for the animal to awaken. δ‐Waves were not apparent at any stage of PB anaesthesia. We applied non‐linear dynamics and information theory to seek evidence of causal relationships between the cardiac, respiratory and slow δ‐oscillations. We demonstrate that, for both groups, respiration drives the cardiac oscillator during deep anaesthesia. During shallow KX anaesthesia the direction either reverses, or the cardio‐respiratory interaction becomes insignificant; in the deep phase, there is a unidirectional deterministic interaction of respiration with slow δ‐oscillations. For PB anaesthesia, the cardio‐respiratory interaction weakens during the second phase but, otherwise, there is no observable change in the interactions. We conclude that non‐linear dynamics and information theory can be used to identify different stages of anaesthesia and the effects of different anaesthetics.

Sex-related difference in collateral sprouting of nociceptive axons after peripheral nerve injury in the rat.

Possible sex-related differences in the extent of collateral sprouting of noninjured nociceptive axons after peripheral nerve injury were examined. In the first experiment, peroneal, tibial, and saphenous nerves were transected and ligated in female and male rats. Eight weeks after nerve injury, skin pinch tests revealed that the nociceptive area of the noninjured sural nerve in the instep skin expanded faster in females; the final result was a 30% larger increase in females than in males. In the second experiment, the end-to-side nerve anastomosis was used as a model for axon sprouting. In addition to the previous procedure, the end of an excised peroneal nerve segment was sutured to the side of the intact sural nerve. Eight weeks later, collateral sprouting of nociceptive axons into the anastomosed peroneal nerve segment was assessed by the nerve pinch test and axon counting. There was no significant difference with respect to the percentages of male and female rats with a positive nerve pinch test. The number of myelinated axons in the anastomosed nerve segment was significantly larger in female (456 +/- 217) than in male (202 +/- 150) rats, but the numbers of unmyelinated axons were not significantly different. In normal sural nerves, the numbers of either all myelinated axons or thin myelinated axons did not significantly differ between the two sexes. Therefore, the more extensive collateral axon sprouting observed in female than in male rats is probably due to the higher sprouting capacity of thin myelinated sensory axons in females.

Interneuronal signalling is involved in induction of collateral sprouting of nociceptive axons

Collateral sprouting of cutaneous nociceptive axons into the adjacent denervated skin critically depends on the nerve growth factor, presumably originating from the degenerated neural pathways and denervated skin. We hypothesised that the degenerated neural pathways are necessary, but not sufficient, to induce collateral sprouting of nociceptive axons, and, in addition, that the interaction between the injured and non-injured neurones within a dorsal root ganglion can trigger sprouting of nociceptive axons also in the absence of the denervated skin. End-to-side nerve anastomosis, made in female Wistar rats by suturing the end of an excised peroneal nerve segment to the side of the intact sural nerve, was used as a model for sprouting which allowed us to study the putative induction mechanisms separately. If the nerves adjacent to the sural nerve were transected concomitantly with the coaptation of the end-to-side anastomosis, robust nociceptive axon sprouting into the anastomosed nerve segment was observed by the nerve pinch test and counting of myelinated axons. Collateral sprouting did not occur, however, either if the cells in the anastomosed nerve segment were killed by freezing and thawing, or if the adjacent nerves had not been injured. However, if the ipsilateral dorsal cutaneous nerves, having their neurones in the same dorsal root ganglia as the sural nerve, were transected, but no other nerves were injured, then the sural nerve axons sprouted in abundance through the anastomosis even in the absence of denervated skin around the sural nerve terminals. From these results we suggest that cells (probably proliferating Schwann cells) in the degenerated neural pathways are necessary but not sufficient to induce collateral sprouting of nociceptive axons, and that interactions between the injured and non-injured neurones within the dorsal root ganglion (i.e. direct or indirect interneuronal signalling) are important in this regard.

Partial fast-to-slow conversion of regenerating rat fast-twitch muscle by chronic low-frequency stimulation.

Chronic low-frequency stimulation (CLFS) of rat fast-twitch muscles induces sequential transitions in myosin heavy chain (MHC) expression from MHCIIb → MHCIId/x → MHCIIa. However, the ‘final’ step of the fast-to-slow transition, i.e., the upregulation of MHCI, has been observed only after extremely long stimulation periods. Assuming that fibre degeneration/regeneration might be involved in the upregulation of slow myosin, we investigated the effects of CLFS on extensor digitorum longus (EDL) muscles regenerating after bupivacaine-induced fibre necrosis. Normal, non-regenerating muscles responded to both 30- and 60-day CLFS with fast MHC isoform transitions (MHCIIb → MHCIId → MHCIIa) and only slight increases in MHCI. CLFS of regenerating EDL muscles caused similar transitions among the fast isoforms but, in addition, caused significant increases in MHCI (to ∼30% relative concentration). Stimulation periods of 30 and 60 days induced similar changes in the regenerating bupivacaine-treated muscles, indicating that the upregulation of slow myosin was restricted to regenerating fibres, but only during an early stage of regeneration. These results suggest that satellite cells and/or regenerating fast rat muscle fibres are capable of switching directly to a slow program under the influence of CLFS and, therefore, appear to be more malleable than adult fibres.

Extent of Nociceptive Dermatomes in Adult Rats Is Not Primarily Maintained by Axonal Competition

Nociceptive innervation territories of individual peripheral and spinal nerves in the skin of the rat hind paw were investigated. In addition, the hypothesis that competitive interactions among the axons from adjacent dorsal root ganglia (DRG) play an important role in maintenance of dermatomal extent in adult animals was tested. The area of innervation territories of individual spinal and peripheral nerves was determined by nociceptive pinch test of the skin after extirpation of adjacent DRGs or transection of adjacent peripheral nerves, respectively. Positions of nociceptive dermatomes and innervation territories of peripheral nerves were similar to the territories innervated by the C-fibers described earlier by dye extravasation technique. In contrast, our results convincingly demonstrated substantial overlap of nociceptive (probably A delta) fibers from adjacent dermatomes in which the autonomous innervation areas were only about one-half of the maximal areas. Nociceptive territories of peripheral nerves overlapped, too. Accordingly, we could find no autonomous innervation area of the sural nerve. Two weeks after extirpation of adjacent DRGs, the area of each of the isolated dermatomes L3, L4, and L5 increased only by about 10%, and it did not change detectably during the next 6 months. The results of our study (a) support the view that innervation fields supplied by the nociceptive (probably A delta) fibers are greater and display more overlap than those supplied by the C-fibers of the same nerve and (b) suggest that axonal competition for innervation territory is not decisive for maintenance of dermatomal borders in the adult rat.

Chapter 26: Age-related differences in the reinnervation after peripheral nerve injury.

Numerous and extensive functional, structural, and biochemical changes characterize intact aged peripheral nervous system. Functional recovery after peripheral nerve injury depends on survival of injured neurons and functional reinnervation of target tissue by regeneration of injured axons and collateral sprouting of uninjured (intact) adjacent axons. The rate of axonal regeneration becomes slower and its extent (density and number of regenerating axons) decreases in aged animals. Aging also impairs terminal sprouting of regenerated axons and collateral sprouting of intact adjacent axons, thus further limiting target reinnervation and its functional recovery. Decreased survival of aged noninjured and injured neurons, limited intrinsic growth potential of neuron, alteration in its responsiveness to stimulatory or inhibitory environmental factors, and changes in the peripheral neural pathways and target tissues are possible reasons for impaired reinnervation after peripheral nerve injury in old age. The review of present data suggests that this impairment is mostly due to the age-related changes in the peripheral neural pathways and target tissues, and not due to the limited intrinsic growth capacity of neurons or their reduced responsiveness to trophic factors. Age-related alterations in the soluble target derived neurotrophic factors, like nerve growth factor, and nonsoluble extracellular matrix components of neural pathways, like laminin, might be important in this respect.

Collateral sprouting of sensory axons after end-to-side nerve coaptation—A longitudinal study in the rat

The end-to-side nerve coaptation is able to induce collateral sprouting of axons from the donor nerve and to provide functional reinnervation of the target tissue. Sensory axon sprouting and its effects on the donor nerve up to 9 months after the end-to-side nerve coaptation were studied in the rat. Peroneal, tibial and saphenous nerves were transected and ligated, and the distal stump of the transected peroneal nerve was sutured to the side of the uninjured sural nerve. The average skin area of the residual sensitivity to pinch due to the axons sprouting through the recipient peroneal nerve did not change statistically significantly between 4 and 9 months after surgery. Axon counting, measurements of compound action potentials and retrograde neuron labeling indicate that the sprouting of the myelinated sensory axons and unmyelinated axons through the recipient nerve was largely completed by 2 months and 4 months after the end-to-side nerve coaptation, respectively, and remained stable thereafter for at least 9 months. A decrease in the amplitude and area of the CAP of myelinated fibers, observed in the donor nerve up to 4 months after surgery, was probably due to mild degeneration of nerve fibers and a tendency of the diameter of myelinated axons to decline. However, no significant changes in functional, electrophysiological or morphological properties of the donor nerve could be observed at the end of the observational period, indicating that end-to-side nerve coaptation has no detrimental effect on the donor nerve on a long-term scale.

The effect of hyperbaric oxygen treatment on early regeneration of sensory axons after nerve crush in the rat

Abstract  The effect of hyperbaric oxygen treatment (HBO) on sensory axon regeneration was examined in the rat. The sciatic nerve was crushed in both legs. In addition, the distal stump of the sural nerve on one side was made acellular and its blood perfusion was compromised by freezing and thawing. Two experimental groups received hyperbaric exposures (2.5 ATA) to either compressed air (pO2 = 0.5 ATA) or 100% oxygen (pO2 = 2.5 ATA) 90 minutes per day for 6 days. Sensory axon regeneration in the sural nerve was thereafter assessed by the nerve pinch test and immunohistochemical reaction to neurofilament. HBO treatment increased the distances reached by the fastest regenerating sensory axons by about 15% in the distal nerve segments with preserved and with compromised blood perfusion. There was no significant difference between the rats treated with different oxygen tensions. The total number of regenerated axons in the distal sural nerve segments after a simple crush injury was not affected, whereas in the nerve segments with compromised blood perfusion treated by the higher pO2, the axon number was about 30% lower than that in the control group. It is concluded that the beneficial effect of HBO on sensory axon regeneration is not dose‐dependent between 0.5 and 2.5 ATA pO2. Although the exposure to 2.5 ATA of pO2 moderately enhanced early regeneration of the fastest sensory axons, it decreased the number of regenerating axons in the injured nerves with compromised blood perfusion of the distal nerve stump.

The contribution of lumbar sympathetic neurones activity to rat's skin blood flow oscillations.

Skin blood flow on the rat’s paws using laser Doppler flowmeter, electrical activity of the heart (ECG) and respiration were measured simultaneously. The signals were recorded for 20 minutes, both before and after denervation, at core temperature 37°C and 38.5°C, that was maintained constant during the recordings. Spinal nerve fibres, at the level L3–L4, were transected. Experiments were performed on 15 adult Wistar rats under general anaesthesia. The oscillations in the measured signals were analysed in the time-frequency domain using wavelet transform. On the frequency region from 0.7Hz to 5Hz two characteristic peaks were observed in the skin blood flow spectrum. They correspond to the main peaks in the spectra of the ECG (around 3.3Hz) and respiration (around 1.3Hz). Several additional peaks were observed in the low frequency region, from 0.01 to 0.7Hz, in all measured signals. In this frequency region the relative energy contribution of the blood flow oscillations decreased after denervation only in the denervated left hind paw. This difference was not statistically significant at 37°C (p=0.098, Kruskal-Wallis test) but became statistically significant at 38.5°C (p=0.017). Relative energy contribution of the low frequency region, from 0.01 to 0.7Hz, decreased 2.5-fold in the blood flow of the denervated paw. Within this region the relative energy contribution decreased significantly in two intervals, from 0.01 to 0.08Hz and from 0.08 to 0.2Hz (p=0.023). In the higher frequency region, from 0.7 to 5Hz, o statistically significant differences were obtained in any paws when compared before and after denervation at the same core temperature. We conclude that the activity of lumbar sympathetic neurones contributes to low frequency skin blood flow oscillations.

Coupling between visual evoked cerebral blood flow velocity responses and visual evoked potentials in migraneurs

Neurovascular coupling may be altered in migraneurs. Therefore, visual evoked potentials (VEP) and visually evoked cerebral blood flow velocity responses (VEFR) were simultaneously recorded in 30 healthy controls and 30 migraneurs interictally using a checkerboard stimulus with visual contrasts of 1%, 10% and 100%. The VEFR were measured in the posterior cerebral artery using transcranial Doppler and VEP were recorded from occipital leads. We found an increase in VEFR and VEP in both the healthy and migraneur groups (P < 0.01). VEFR were significantly higher in migraneurs (P < 0.01), while VEP did not significantly differ between the groups (P > 0.05). Regression showed a significant association between VEP and VEFR in both healthy controls (r = 0.66, P < 0.01) and migraneurs (r = 0.63, P < 0.01). The regression coefficient of migraneurs (b = 0.88, SE = 0.08) was significantly higher than that of healthy controls (b = 0.55, SE = 0.07) (P = 0.04). We conclude that neurovascular coupling is increased in migraneurs interictally.