Microscopio electrónico de barrido (MEB)

GABAergic agents Local anaesthetics Analgesic agents Anaesthesia NMDA antagonists Opioids NMDA antagonists NSAIDs Adrenergic agents'] Others: e.g. cannabinoids low dose local anaesthetics purinergic antagonists WÈsitizati educed peri Unconsciousness Amnesia reduced muscle tone Autonomic depression increased withdrawal reflex threshold Analgesia Reduced hyperalgesia Diagram 11

The common classes of anaesthetic and analgesic drugs are listed here together with a list of physiological alterations that can be used to define each pharmacological state. In this scheme, an increase in reflex withdrawal threshold is classed as an anaesthetic effect while analgesia is defined specifically as a reduction in hyperalgesia.

3 .5 .3 N itrous O xide: efficacy during developm ent Nitrous oxide has been in clinical use for over 150 years. It is used as a supplement and carrier gas for typical volatile anaesthetic agents and to reduce

requirements of the latter. It is thought to have significant analgesic actions of its own that make it a useful either when used alone or as an adjunct to other analgesic agents in particular clinical situations.

Debate about the mechanism of action of this widely used gas continues. Though several molecular targets have been identified, two dominant hypotheses share current favour. One of these suggests that nitrous oxide causes the release of opioid peptides from midbrain neurons (Maze and Fujinaga, 2000). Importantly, this theory predicts that N 20 will have no antinociceptive action prior to the maturation of

descending noradrenergic pathways (4 weeks in rats and toddler age in humans). This has obvious implications for paediatric anaesthetists as the conjecture that N 20 does not provide any measurable analgesia in human infants under two years old would be quite surprising.

Much of the data supporting the opioid/noradrenergic theory rely heavily on the tail flick latency test in rats, a test which models acute nociception rather than post­ injury hypersensitivity. Significantly, the data preclude a direct action of N 20 on spinal nociceptive neurons(Zhang et al., 1999; Guo et al., 1996). It is difficult to resolve these data with the now well accepted action of nitrous oxide on NMDA receptors (Jevtovic- Todorovic et al., 1998)- given that a) spinal nociceptive processing does involve NMDA receptor mediated transmission (Woolf and Thompson, 1991); b) that NMDA receptor antagonists such as ketamine and MK-801 do have significant antinociceptive actions at subanaesthetic doses and that these actions are at least partially mediated at a spinal level (Hao et al., 1998; Dickenson and Sullivan, 1990) and c) that the kinetics of nitrous oxide do not preclude a spinal cord biophase (i.e. there is no reason to expect spinal cord tensions to be less than cerebral tissue tensions) (Stenqvist, 1994) - a direct antinociceptive action of nitrous oxide at a spinal level would be predicted. The

experiments in which antinociceptive actions of nitrous oxide were abolished by supraspinal intervention require careful re-interpretation.

Perhaps a key to this re-interpretation is the distinction between acute

antinociception and anti-hyperalgesia. For nitrous oxide, the first may depend on an opioid/noradrenergic mechanism while the latter may be mediated by NMDA receptor blockade.

Data on the efficacy of nitrous oxide in early developmental stages are critically important for the continued rational use of the agent in paediatric anaesthetic and analgesic practice. Data confirming a spinal site of action for N 20 in rat pups prior to

the maturation of descending noradrenergic pathways would argue against the importance of the opioid/noradrenergic hypothesis. The data presented here while confirming efficacy of the gas in early development stages, require further development before they can be used in the debate outlined above. Experiments testing the efficacy of nitrous oxide in carrageenan inflamed pups and its efficacy in spinally transected rat pups will prove more valuable.

In view of the data confirming an interaction between nitrous oxide and glutamatergic neural transmission- in particular, the NMDA receptor system, an anaesthetic regime that did not include this agent (i.e. Oxygen/Air/halothane) was used for all pharmacodynamic studies of antinociceptive agents.

3.5.4 U rethane an aesth esia

The trials involving the use of urethane revealed three preliminary findings: a) that the anaesthetic dose in 21 day old rat pups (approx. 2g/kg) is greater

than that typically reported for adult pups (l-1.25g/kg).

b) that urethane dose-dependently inhibits the withdrawal reflex as measured electromyographically and this relationship is defined by a very steep dose- response curve between 1.7 and 2.5 g/kg.

c) following peripheral inflammation, sensitivity of reflex excitability to urethane anaesthesia appears to be increased.

The age related differences in effective dosage may be the result of kinetic factors (e.g. slower absorption, an altered volume of distribution or greater clearance in younger pups) or dynamic factors reflecting age related changes in neurotransmission (in particular, the receptor-ligand systems on which urethane may act). No data are available regarding the kinetics of urethane in early development and there is no consensus yet regarding the exact site of action of the drug. Little further comment is possible.

The steep dose-response relationship between urethane and reflex

responsiveness imply that this form of anaesthesia is innappropriate for the study of epidurally administered analgesic drugs in this model.

The surprising finding of increased sensitivity to urethane in the face of peripheral tissue inflammation- if not a spurious finding, is open to speculative explanation. The finding suggests that urethane may act on a mechanism that is more prominent /up regulated in the inflammatory state e.g. the NMDA receptor system or the sP peptidergic system. Both of these possibilities await clearer definition of the mechanism of action of urethane. Another difference between the naive and inflamed state is the activation of the sympathetic-pituitary-adrenal axis. Urethane is known to

activate this important homeostatic system (Reinert 1964). This system may have significant inhibitory effects on nociceptive processes via release of “stress hormones”. An increased sensitivity to urethane may then conceivably be due to greater stress response in pups that have been “primed” by a prior inflammatory insult. This idea has parallels with the finding of tachyphylaxis to carrageenan described by Battacharya (Battacharya et al 1987).

Further study of these issues may allow clearer interpretation of much

experimental data already gathered from animal models in which urethane anaesthesia has been utilised.

Chapter 4 MODELS OF INFLAMMATION AND

INFLAMMATORY PAIN

4.1 Inflammation- a gen eral o v e r v i e w

4 .1 .1 D efin ition

Inflammation is a coordinated tissue response to cellular debris and

substances or antigens recognised as foreign to the organism. The response may be either localised or systemic. Local inflammation, which is the focus of this discussion, is defined in clinical practice by the appearance of redness, induration, heat and pain. To these

original features of Celsus (30BC), Virchow added “loss of function” of the inflamed tissue (usually implying a loss of movement in an inflamed limb).

Histologically, it is characterised by vascular dilatation, pavementing and then migration of leucocytes and an increase in tissue fluid (inflammatory exudate)

Physiological processes involved include: vasodilation and oedema formation (exudation), chemotaxis, leucocyte degranulation, phagocytosis by neutrophils and sensitization of peripheral nerves. A later proliferative process precedes the resolution of inflammation (Walter, 1987).

Central in the initiation and control of the response is the immune system. Activation of the response can be achieved through

a) degranulation of mast cells residing within extravascular tissue spaces releasing histamine and other mediators (NGF, histamine and 5HT). On activation, mast cells are able to metabolise arachachidonic acid to mediators such as the leukotrienes, prostaglandins and thromboxanes.

b) leucocyte-endothelial adhesion with subsequent release of lysosomal compounds.

c) the release of arachadonic acid metabolites from damaged cells notably, endothelial cells.

d) enzyme cascade activation, in particular activation of Hageman factor (FXII) eventually leads to the activation of the coagulation, kinin, fibrinolytic and complement systems.

Endothelial cell and mast cell reactions are probably the crucial steps in the triggering of most inflammatory responses (Underwood, 2000; Walter, 1987).

4 . 1 . 2 T ypes

Inflammation is most usually classified into acute and chronic forms. Chronic inflammation differs not only in its time frame but more importantly in the molecular and cellular processes that underlie it. An older classification into “phagocytic and non-phagocytic” forms was probably just a reflection of the division between the pattern of acute and chronic inflammation. The following discussion is focused on acute inflammation.

The entire acute inflammatory response is characterised by co-operativity between inflammatory and immune cells together with a large latent potential. This is represented by the storage and sequestering of cells and mediators and the plasma store of inactive

precursors. Once triggered, the process is sequential and involves several powerful amplification systems whose spatial and temporal spread are under constant control by inhibitory mechanisms. The process therefore reflects a changing equilibrium between often competing processes (Underwood, 2000).

Some of these characteristics are shared by the nervous system. This observation may be more than just incidental as a close inter-relationship between the CNS and the immune system is being increasingly recognized (Watkins and Maier, 1999; Perry and Gordon, 1997). Interaction between these two systems is bi-directional and especially evident in what is now termed the “acute phase response” or “sickness response” (Salzet et al., 2000).