Leukocytes

Leukocytes – (White Blood Cells)

Leukocytes can be classified as accordingly:

Cell Type          Abundance (%) Diameter (µm)

Neutrophils      60-70                    12-15

Eosinophils       2-4                         12-15

Basophils          0-1                         12-15

Lymphocytes    20-30                      6-18

Monocytes       3-8                          12-20

(Cells in red are agranular, they are agranulocytes. Cells in blue are granular, they are granulocytes)

Leukocytes leave blood capillaries by passing between endothelial cells and penetrating into the connective tissue by a process known as diapedesis. Diapedesis is increased in individuals infected with micro-organisms.

Neutrophils (Polymorphonuclear Leukocytes)

Neutrophils constituting around 60-70% of the total circulating leukocytes and they have a diameter of 12-15µm. Neutrophils are polynuclear in appearance however these lobes are in fact joined. (May have 2-5 lobes however, 3 seems average). Neutrophils are also granular.

Neutrophils are adept at surviving in aerobic conditions, making them useful for killing bacteria and cleaning up debris in low oxygenated environments e.g. inflamed or necrotic tissue.

Neutrophils are relatively short lived with a life span of only 1-4 days, where-after they die by apoptosis.

Neutrophils act as a defence against micro-organism invasion, especially bacteria. They are active phagocytes of small particles and are sometimes known as microphages to distinguish them from macrophages which are larger cells.

Bacteria adhere to neutrophil surfaces, where they are phagocytosed. They then occupy vacuoles within the cell (phagosomes). The granules fuse with the phagosomes releasing their content – enzymes which kill and digest the micro-organisms

Eosinophils

Eosinophils are much less numerous than neutrophils (2-4%). They are characterised by their bilobed nucleus and the high affinity their granules have for eosin.

The role of eosinophils in the immune system is to fight against parasitic organisms (Eosinophils are of much higher concentrations during parasite attacks and allergic reactions). They secrete digestive enzymes directly onto the parasite which may also result in localised tissue damage.

Basophils

Basophils make up less than 1% of the total blood leukocyte count; they are therefore rare to find in blood smears. Their nuclei are divided into irregular lobes which are hard to distinguish because of the overlying granules.

Basophils contain specific granules which themselves contain heparin and histamine, they are capable of producing leukotrienes (fatty molecules which are used as paracrine/autocrine signalling molecules), these leukotrienes can cause slow contractions of smooth muscle.

It is believed that basophils supplement mast cell function in immediate hypersensitivity reactions by migrating into the connective tissues.

Lymphocytes

Lymphocytes can be classified into two primary groups (depending on their specific surface receptor), either T or B lymphocytes. Both T and B lymphocytes possess specific antigen receptors on their surface, which is important in immunological responses (see later articles for immunology and lymphocytes)

Lymphocytes vary greatly in lifespan, from a few days to years.

Lymphocytes are also the only leukocytes which can return from tissues back to the blood after diapedesis.

Monocytes

The nuclei of monocytes are generally oval, horseshoe or kidney shaped. Because of the delicate chromatin distribution in monocytes they stain lighter than the larger lymphocytes – aiding identification

After crossing capillary walls and entering connective tissues, monocytes differentiate into macrophages

Platelets

Platelets (thrombocytes) are non-nucleated cell fragments 2-4µm in diameter (the small specs in the picture.) They originate from fragmentation of megakaryocytes residing in the bone marrow. The primary role of platelets is the promotion of blood clotting and the repair of gaps in the walls of blood vessels.

The Role of Platelets in the Clotting Cascade

1.Primary Aggregation: Damage to the endothelium of blood vessels causes absorption of plasma proteins on the subjacent collagen. Platelets immediately begin to aggregate on this damaged tissue forming a platelet plug.

2.Secondary Aggregation: Platelets in the plug release the contents of their granules, ADP s a potent inducer of platelet aggregation.

3.Blood Coagulation: Factors from platelets, damaged blood vessels and blood plasma promote a cascade of approximately 13 proteins which give rise to the formation of fibrin. Fibrin forms a 3D network of fibres which trap red blood cells leukocytes and platelets to form a blood clot.

4.Clot Retraction: This is when the formed clot which bulges into the blood vessel begins to contract. This is due to the interaction of platelet actin, myosin and ATP.

5.Clot Removal: The blood vessel wall is repaired by new tissue formation. The clot is then removed by the enzyme plasmin.

Recommended further reading on this subject, specifically the platelet plug formation and the clotting cascade can be found at:

http://www.blobs.org/science/article.php?article=14

This document can be downloaded at http://dl.dropbox.com/u/2561295/Website/Leukocytes.docx

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Non-Steroidal Anti-inflammatory Drugs (NSAIDs)

Introduction

NSAIDs are non-narcotic analgesics (An analgesic reduces or removes the sensation of pain), they are also anti-pyretic (fever) and anti-inflammatory. These effects are produced by the inhibition of the fatty acid cyclooxygenase (COX) which inhibits prostaglandin synthesis.

Because NSAIDs are non-narcotic they do not cause any largely noticeable effects on the CNS (central nervous system) function. This makes them ineffective against normal nociceptive tests, these are test designed to test pain responses in living organisms and they are specifically used in the testing of new analgesic drugs. Methods usually involve the applying of pressure to a specific point of the organism. NSAIDs only raise the pain threshold when pressure is applied to a swollen and inflamed joint (this is known as analgesia via peripheral mechanisms). Therefore NSAIDs are considered anti-inflammatory agents with a mild central analgesic effect (associated with anti-pyretic action). NSAIDs are therefore primarily used in the treatment of acute or chronic conditions producing mild-moderate pain, especially involving the musculo-skeletal system. Principal utilisation occurs in the horse and dog.

Another benefit of NSAIDs non-narcotic function is that, by having an unnoticeable effect on the CNS they can therefore be used as a pre-med drug, before general anaesthetic, without fear of overloading the CNS. The use of an analgesic before the introduction of pain means that a lower dose will be required when pain is inflicted – thus reducing the chances of side effects associated with high doses. However, the type of NSAID must be selected for carefully as there may be a possibility of renal damage/toxicity. There are only a couple of NSAIDs which are believed to be renal safe.

Prostaglandins and Inflammation

NSAIDs work by inhibiting prostaglandin synthesis by targeting the COX enzyme. Prostaglandins activate the inflammatory response giving the production of pain and fever, they are produced when leukocytes reach a site of damaged tissue in an attempt to minimise tissue destruction.

Prostaglandins are involved in several other organs such as the gastrointestinal tract (inhibit acid synthesis and increase secretion of protective mucus), increase blood flow in kidneys, and leukotrienes which promote constriction of bronchi associated with asthma.

Question:

What is/are the precursor(s) of prostaglandins?

The answer may be found in the ‘Mechanisms of NSAID Action’ section.

Cyclooxygenase Enzymes (COX)

COX forms two isoforms, COX-1 and COX-2:

  • COX-1 is often thought of as being the ‘good’ COX; this is due to its involvement in tissue homeostasis. It is required to keep the body ‘normal’ – primarily the synthesis of prostaglandins responsible for protection of the stomach lining. (Constitutive physiological).
  • COX-2 therefore is thought of as the ‘bad’ COX; this is because it is produced during inflammation, by the inflammatory cells which have been activated by cytokines. (Inducible physiological).
  • There is some evidence for a COX-3, a possible variation of COX-1 which is also associated with the inflammatory response. It has been found in the CNS and is affected by paracetamol.

Mechanism of NSAID Action

The primary action of NSAIDs is the inhibition of the COX enzyme, by inhibiting this enzyme the production of prostaglandins are also inhibited. The COX enzyme synthesised prostaglandins from fatty acids such as arachidonic acid.

Most NSAIDs inhibit both major forms of the COX enzyme, however all are still considered toxic. Newer drugs which are believed to be COX-2 specific (thereby not affecting the COX-1 enzyme and allowing prostaglandins associated with normal function to continue normal operation) are relatively safer in chronic use. There are fewer side effects which is what makes them be suited to prolonged periods of use. Examples: Merck’s rofecoxib and etoricoxib, Pfizer’s celecoxib and valdecoxib.

The NSAIDs selective for COX-2 are now however under scrutiny, due to reports of cardiovascular toxicity. These include strokes and myocardial infarctions. This has resulted in the withdrawal of certain COX-2 selective drugs such as rofecoxib. Further studies are suggesting that the cardiovascular toxicity of these COX-2 selective NSAIDs may actually not be much greater than ‘trusted’ NSAIDs such as ibuprofen. However, their toxicity still remains a point of research and discussion.

Other Actions of NSAIDs

Besides from the inhibition of the COX enzyme, other actions include:

  • Inhibit superoxides (toxic) and free radicals
  • Inhibit Bradykinin production (A Peptide which dilates blood vessels, lowering blood pressure)
  • Stabilises lysosomes
  • Inhibits metalloproteinases (Proteolytic enzymes whose catalytic mechanism involves a metal)
  • Antagonises interleukin-1 (fever inducer and controlling factor of lymphocytes) and tumour necrosis factor (TNF – cytokine involved in the induction of inflammation and apoptosis, dysfunction of this factor is believed to be involved with the production of cancers.)

IC50

This is the half maximal inhibitory concentration (IC50). It is a measure of the effectiveness of a compound (typically a drug candidate) in inhibiting biological or biochemical function. This quantitative measure indicates how much of a particular drug is needed to inhibit a given biological process (or component of a process, i.e. an enzyme, cell, cell receptor or microorganism) by half. It is commonly used as a measure of antagonist drug potency in pharmacological research.

This can be related to inhibition of COX enzymes, we can use it to find out how many times a dose required to inhibit COX-1 we need to administer in order to inhibit COX-2, i.e. COX-2/COX-1,  a lower ratio is better as it shows that the drug has a higher selectivity for COX-2 the ‘bad’ COX associated with inflammatory responses.

COX-1 Selective               Drugs                       Amount times dose required to inhibit COX-2

  • Aspirin                                                                  170
  • Piroxicam                                                             250

Less Selective COX-1 Drugs

  • Paracetamol                                                            7
  • Ibuprofen                                                                15

Equipotent

  • Naproxen                                                                  1

COX-2 Selective Drugs

  • Meloxicam                                                               <1

The Anti-Pyretic Effect of NSAIDs

NSAIDs do not affect normal body temperature only when pyrexia (fever) is present, do they alter temperature. Bacterial or endogenous (substances from within the body) pyrogens can act directly on the hypothalamus. Heat temperature is regulated in the hypothalamus by controlling temperature regulating peripheral mechanisms such as vasoconstriction/dilation, sweating, shivering and metabolic activity.

Pyrogens activate hypothalamic COX, increasing prostaglandin concentration. The effect of this is that the set body temperature (Average 37oC) is increased (Pyrexia is considered >38oC). NSAIDs counter this by inhibiting prostaglandin synthesis by the inhibition of COX enzymes. By doing this they have effectively blocked the action of the pyrogens on the CNS and return the raised set body temperature back down to normal levels (37oC).

Commonly Used NSAIDs

Some of the most commonly found NSAIDs and their properties:

Aspirin (Acetylsalicylic acid)

Aspirin is a potent anti-inflammatory drug with mild central analgesic and antipyretic actions. It is administered orally and readily absorbed from the stomach and small intestine, an acid drug is well absorbed in an acidic environment. It is metabolised by tissue /plasma esterases. Aspirin may also be used in low doses, daily to prevent platelet aggregation.

In a healthy body, thromboxane and prostacyclin (eicosanoids – fatty acid signalling molecules) are balanced. Aspirin however disrupts this balance in the favour of prostacyclin, inhibiting aggregation.

Aspirin irreversibly binds to platelet COX, however as platelets are produced every few days, the condition is not permanent, and this is why chronic dosing may be necessary.

Aspirin is toxic in cats due to their lack of the enzyme UDP-glucuronyl transferase, therefore when giving aspirin to cats the maximum stated dose is 25mg/kg daily (Compared to 25mg/kg 3-4 times a day in dogs) Toxicity effects may still appear even at lower doses e.g. vomiting, abdominal pain, anorexia and gastric ulceration.

Paracetamol

Paracetamol is a weak anti-inflammatory drug; however it does have a more potent central analgesia and anti-pyretic effects than aspirin (See IC50 table to see a smaller dose is required of paracetamol to inhibit COX-2, the COX enzyme responsible for inflammatory responses).

Due to paracetamol being well tolerated, producing less gastric irritation than aspirin and having much fewer side effects than aspirin, it has become a predominant household analgesic. Acute overdoses can cause fatal hepatic damage; early symptoms include anorexia, vomiting, diarrhoea and abdominal pain. It is the reactive metabolites of paracetamol latching onto -SH groups that cause hepatic toxicity.

The dog is more resistant to paracetamol toxicity than cats, an oral dose is recommended every 6 hours of 25-30mg/kg.

Phenylbutazone

Phenylbutazone is the most widely used NSAID in equine medicine; however it is extremely toxic in humans. It has a long  t1/2 (half-life) of 70 hours and produces severe gastric ulceration and agranulocytosis. It can be administered orally and by intravenous injection. Due to the acidity of the drug, it is readily absorbed from the stomach/duodenum. Phenylbutazone metabolites are weak acids and therefore preferably excreted in alkaline urine. Training horses may have acidic urine, and so it is recommended not to take Phenylbutazone within 8 days of competition.

Half-life in dogs of Phenylbutazone has been recorded at 3-8 hours (however this may vary dependent on the dose). Phenylbutazone can inhibit the synthesis of prostaglandin in inflammatory exudates for 12-24 hours, with the response lasting for up to three days after the final dose in the course.

Signs of Phenylbutazone toxicity include inappetance and depression with weight loss and oedema. The oedema (fluid retention) is due to the decreased NaCl excretion.

Dosing of Phenylbutazone should not exceed:

  • Day 1 – 4.4 mg/kg twice a day
  • Day 2-4 – 2.2 mg/kg twice a day
  • Day >5 – 2.2 mg/kg daily

Phenylbutazone is also administered in combination with other drugs for the treatment of musculoskeletal disorders e.g. Tomanol – Phenylbutazone and Isopyrin

Meclofenamic Acid (Arquel)

Meclofenamic acid is a potent anti-inflammatory, anti-pyretic analgesic. It is more potent than aspirin but similar in effect. As well as inhibiting COX enzymes it has found to be a prostaglandin antagonist, interacting with prostaglandin receptors. It therefore prevents the action of prostaglandin already present possibly exerting a more rapid reduction of inflammation.

Half-life is 6-8 hours; therapeutic levels are maintained with daily doses.

Unlike other NSAIDs onset of Meclofenamic acid is relatively slow, taking 36-96 hours for effects to begin

Prolonged administration in the horse may lead to anorexia, depression, ulceration of buccal mucosa, gastric haemorrhage or diarrhoea

Naproxen

Naproxen is a propionic acid derivative (like ibuprofen or Ketoprofen) with a tendency for reduced frequency of serious side effects at therapeutic doses. In the horse, half-life of the drug is 5 hours; double daily doses have been proven effective in soft-tissue inflammatory conditions such as myositis. In the dog however, half-life is much longer at around 70 hours, it is therefore recommended to avoid this NSAIDs due to excessive toxicity

Ketoprofen

Ketoprofen is a potent COX inhibitor which is also able to stabilise lysosomal membranes and is a Bradykinin antagonist. It is also reported to be an inhibitor of the lipoxygenase enzyme system (iron-containing enzymes which catalyse the dioxygenation [incorporation of two oxygen atoms] of some polyunsaturated fatty acids). It is around 50x more potent than Phenylbutazone, however this is not accompanied by an equivalent increase in toxicity. It is also suggested that it may be cartilage sparing, neither accelerating chondrocyte damage nor reducing proteoglycan production.

Carprofen

Carprofen is a potent anti-inflammatory drug, but is a weak inhibitor of COX. Its mode of action is not yet known but it significantly inhibits neutrophil migration. Due to weak inhibition of COX, toxicity of Carprofen its toxicity tends to be low.

Piroxicam

Piroxicam is a potent and long lasting anti-inflammatory drug. Its half-life of around 60 hours enables lower dosing (alternate days). A higher frequency of dosing will produce standard NSAID toxic effects (see below, section ‘Side Effects of NSAIDs’)

Meloxicam

Meloxicam is a similar drug, but with a shorter half-life (30-40 hours). It is thought to have greater potency for COX-2 than COX-1 therefore side-effects may be less. It is also thought to be chondroprotective (The slowing of degradation of articular cartilage)

Flunixin

Flunixin is a potent versatile anti-inflammatory drug with a short half-life in all species (2-8 hours). However its duration of action is relatively long (24-36 hours)

When to Use NSAIDs

  • Mild to moderate inflammatory lesions and associated pain
  • Acute inflammation and pain
  • Joint inflammation and pain
  • Suppression of pulmonary oedema
  • Endotoxaemia
  • Anti-thrombic

Side Effects of NSAIDs

  • Gastric irritation and ulceration – This is a main side effect of chronic NSAID use, it occurs because when NSAIDs inhibit the COX-1 enzyme, COX-1 synthesises prostaglandins associated with inhibiting acid synthesis and increasing secretion of protective mucus. SO by inhibiting COX-1, the stomach becomes unprotected from the gastric acid causing irritation and possibly ulceration in chronic use.
  • You can protect the gut by administering proton pump inhibitors (Losec), prostaglandin analogues (Misoprostol) or H2 receptor antagonists (Zantac)
  • Vomiting and diarrhoea
  • Hepatotoxicity
  • Renal papillary necrosis, chronic nephritis
  • Bone marrow disturbance
  • Skin rashes
  • Respiratory distress

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