The Genitourinary Tract The Renal System

Probably the most intense of pains experienced by humans is that resulting from nephrolithiasis (kidney stones), which can be reproduced with some symptomatic accuracy in rats. With any pain model, but especially those of a potentially severe nature, the ethical implications should be addressed. A stimulus that cannot be terminated by either the experimenter or animal (e.g., by escape or an operant response) is of particular concern, and a factor that is more common in visceral than somatic pain models due to the nature of the pain. With this in mind, a number of models have been introduced to replicate nephrolithiasis, one of which is the introduction of artificial ureteral calculosis in the rat—the surgical formation of an artificial kidney stone in the ureter. Such a model was presented by Giamberardino et al. (74), and involves the injection of 20 p.L dental resin cement solution into the upper-third of one ureter. The cement will harden and block the ureter, resulting in a marked hyperalgesia that is accompanied by visceral pain-related behavior (Fig. 6) not seen in sham-operated rats or rats submitted to a permanent ligature of one ureter (74,75). The threshold for vocalization to

0 3 S 9 12 15 18 21 24 27 Saline Butyrate 200 mM

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0 3 S 9 12 15 18 21 24 27 Saline Butyrate 200 mM

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Figure 5 (See color insert) Intracolonic butyrate produces enhanced colonic sensitivity and referred hypersensitivity in the rat. The effect of six (twice daily) intracolonic infusions of 1 mL saline or butyrate solution (8, 40, 200, or 1000 mM) on the pressure thresholds inducing a specific behavior following colorectal distension (A), and on forces exerted by application of von Frey filaments to the lumbar abdominal skin required to induce a reaction (C). These effects are significantly enhanced in female compared to male rats (B and D). Source: From Ref. 56.

0 3 6 9 12 15 10 21 24 27 saline Sulyrate 200 mM

Bulyrate Time (days)

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Figure 5 (See color insert) Intracolonic butyrate produces enhanced colonic sensitivity and referred hypersensitivity in the rat. The effect of six (twice daily) intracolonic infusions of 1 mL saline or butyrate solution (8, 40, 200, or 1000 mM) on the pressure thresholds inducing a specific behavior following colorectal distension (A), and on forces exerted by application of von Frey filaments to the lumbar abdominal skin required to induce a reaction (C). These effects are significantly enhanced in female compared to male rats (B and D). Source: From Ref. 56.

electrical stimulation of the obliquus externus muscle is decreased as the number and duration of visceral episodes increases (75). This observation may be used as a correlate of referred pain, a phenomenon that is often found in human visceral pain disorders, as is the finding that ureteral pain sensitivity varies with the estrous cycle in both humans and rats with nephro-lithiasis (76). These similarities add weight to the rationale for the use of such an animal model. (It is important to note, however, that rodent vocalizations at ultrasonic frequencies are better related to painful experiences than those vocalizations at frequencies within the human aural range.) However, while useful for behavioral analysis of visceral pain, this model is not ideal for the study of neuronal activation, as the precise time at which the experimental stone forms is unknown.

An alternative model is the percutaneous ureteral obstruction model, proposed by Avelino et al. (77). Following a low-midline abdominal incision (under halothane anesthesia), a knot is loosely tied around one ureter using nylon, the ends of which are exteriorized before the wound is closed, and the animal allowed to recover. Eight days later (a time point at which spinal c-fos expression, evoked by the surgical procedure, is completely gone (77), and thus only the effect of the ureteral obstruction is understudy). The ends of the nylon thread can be pulled to ligate the ureter. Rats that have undergone this procedure show pain-like behavior, but only after the knot has been pulled tight (77). Note, however, that previous studies that employed an ureteral ligature found no significant muscle hyperalgesia as determined by the vocalization threshold to stimulation of the obliquus externus muscle (74).

The pain produced from models such as these (especially from ureteral ligation) may result from distension of the ureter central to the ligation or the renal pelvis. Distension pressures greater than 25 mmHg in the rat produce cardiovascular pseudaffective responses likely to represent painful experiences (78), a claim strengthened by the observation that pressor responses to ureter distension are significantly, and dose-dependently, reduced following

Figure 6 Behavioral phases of a visceral episode. Rats with implanted ureteral stones present graded visceral behavioral episodes as reported by Giamberardino et al. (75). These behavioral manifestations consisted of: (A) hump-backed position; (B) licking of the lower abdomen, the left flank, or both; (C) contraction of the left oblique musculature with inward moving of the hind limb; (D) stretching of the body; (E) squashing of the lower abdomen against the floor; (F) supine position with left hind limb adducted and compressed against the abdomen. Source: From Ref. 75.

Figure 6 Behavioral phases of a visceral episode. Rats with implanted ureteral stones present graded visceral behavioral episodes as reported by Giamberardino et al. (75). These behavioral manifestations consisted of: (A) hump-backed position; (B) licking of the lower abdomen, the left flank, or both; (C) contraction of the left oblique musculature with inward moving of the hind limb; (D) stretching of the body; (E) squashing of the lower abdomen against the floor; (F) supine position with left hind limb adducted and compressed against the abdomen. Source: From Ref. 75.

intravenous morphine (78). Again, this model is not appropriate for behavioral studies of ureter-evoked visceral pain, as the animals are both anesthetized and under neuromuscular block. Distension of the renal pelvis in anesthetized rats produces a depressor response (a decrease in blood pressure that is restored following cessation of the distension stimulus) that is reversed by morphine administration (79).

Rather than inserting an artificial kidney stone into the ureter, other models have been developed to promote the "natural" formation of Ca2+ stones. The rat model of calcium oxalate nephrolithiasis (the hyperoxaluric rat, hyperoxaluria being excessive urinary excretion of oxalate, and calcium oxalate stones are the most common in clinical nephrolithiasis) involves the inclusion of 1% ethylene glycol in the drinking water and the subsequent formation of calcium oxalate deposits in the proximal tubules of the kidney (80). Enhanced calcium oxalate deposition was reported in animals given ethylene glycol while on a Mg2+-deficient diet (80,81). A subsequent study used ethylene glycol with 1% ammonium chloride to produce nephrolithiasis, and this group have published the experimental conditions required to achieve different phases of calcium oxalate nephrolithiasis (82). Alternatively, there is a genetic model of hypercalciuria (excessive urinary calcium excretion), a condition that is common in patients with calcium oxalate nephrolithiasis, known as the genetic hypercalciuric rat. Hypercalciuria occurs spontaneously in a small population of rats (83), and these rats can be inbred to establish an experimental colony (84). Unfortunately, however, there are no behavioral data to indicate whether these animals exhibit behaviors suggestive of visceral pain.

Recently, two new rat models of calcium oxalate nephrolithiasis have been reported: the first, an example of a surgical intervention—small-bowel resection (85); the second, pharmacological—using a selective cyclooxygenase 2 inhibitor (86). Both these models also involve dietary modification, and it will be interesting to see if these models exhibit behavioral pain responses.

51 Tips for Dealing with Kidney Stones

51 Tips for Dealing with Kidney Stones

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