Hypertrophic neurons innervating the urinary bladder and colon of the streptozotocin-diabetic rat

277

Brain Research, 609 (1993) 277-283 © 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00

BRES 18752

Hypertrophic neurons innervating the urinary bladder and colon of the streptozotocin-diabetic rat Irving N a d e l h a f t a

a,b,c, P e d r o

L. V e r a a,b,c a n d B e r n a r d S t e i n b a c h e r

a

VA Medical Center, Departments of b Neurological Surgery and c Pharmacology, University of Pittsburgh Medical School, Pittsburgh, PA 15213 (USA) (Accepted 17 November 1992)

Key words: Bladder; Diabetes; Diuresis; Hypertrophy

Female rats were made diabetic with an intravenous injection of streptozotocin (STZ) producing bladder hypertrophy. Using fluorescent dyes injected into the bladder or the colon, we have measured the size of neurons in various ganglia associated with these organs in control and STZ-diabetic rats. These include (1) postganglionic neurons in the pelvic ganglion, (2) postganglionic neurons in the inferior mesenteric ganglion, (3) dorsal root ganglion neurons, (4) sympathetic chain ganglion neurons, (5) preganglionic neurons in the sacral parasympathetic nucleus, (6) motor neurons in Onuf's nucleus innervating the external urethral sphincter. In addition we have measured neurons in some of these groups for rats which have been maintained on a 5% sucrose in water and restricted food diet. In the STZ-diabetic animals only those neurons which make direct contact with the bladder or the colon were found to be hypertrophied (15-70%). In the diuretic animals, only neurons directly innervating the bladder exhibited hypertrophy. We speculate that atrophic factor transported from the organ to the neuron is responsible for this effect.

INTRODUCTION

Hypertrophy of smooth muscle occurs as a consequence of various stresses placed upon the organ (e.g. obstruction, denervation, stretch; for a review see Gabella6). Urinary bladder hypertrophy is often a consequence of diabetic neuropathy in humans 3'5'14 and is observed routinely in streptozotocin (STZ)-diabetic rats 11'16't8'22. In the case of urinary bladder hypertrophy following urethral obstruction 23 a concomitant observation was a substantial increase in the sizes of bladder postganglionic neurons located in the major pelvic ganglia. This finding suggested that the urinary bladder hypertrophy observed in the STZ-diabetic rat might also be accompanied by changes in those neurons concerned with micturition. In the experiments reported here we have measured the size of neurons in various ganglia associated with micturition and defecation in control and STZ-diabetic rats. These include (1) bladder postganglionic neurons in the pelvic ganglion, (2) postganglionic neurons innervating the bladder and the colon in the inferior mesenteric ganglion, (3) dorsal

root ganglion neurons innervating the bladder and the colon, (4) sympathetic chain ganglion neurons innervating the bladder and the colon, (5) preganglionic neurons in the sacral parasympathetic nucleus 15, (6) motor neurons in Onuf's nucleus 12 innervating the external urethral sphincter. In addition we have measured neurons in some of these groups for rats which have been maintained on a 5% sucrose in water and restricted food diet. Bladder hypertrophy has been reported in this latter group without any evidence of hyperglycemia11. MATERIALS AND METHODS Twelve female Sprague-Dawley rats (average weight: 205 + 29 g) were used in this study. Seven were made diabetic with streptozotocin (STZ, 60 mg/kg) by injection into a tail vein of a solution containing 60 mg of STZ per ml of 0.01 M citrate-buffered saline. Two of these animals died before they could be used in experiments. Five age-matched rats were used as controls. All these animals were individually housed with access to food and water ad libitum. Two additional animals were placed on a special diet (5% sucrose in drinking water plus 10 g of rat chow per day) to produce diuresis. The plasma level of glucose was measured about a week after the injection of STZ by a blood glucose monitor (Lifescan). The glucose

Correspondence: I. Nadelhaft, VA Medical Center, University Drive C, Pittsburgh, PA 15240, USA. Fax: (1) (412) 683-4917.

278 levels in units of m g / d l were: diabetics: 344.7±51.9, controls: 127.0 ± 14.6 (mean_+ S.E.M.). Glucose levels in the sucrose-fed rats (i.e. diuretic rats) was measured at the time of sacrifice (1 month after starting the special diet) and were 142 and 166 m g / d l . To label the various groups of neurons to be studied, the animal was anesthetized with halothane (1.5% in oxygen at 1 l / m i n ) and the abdominal cavity opened by a midline incision exposing the urinary bladder, urethra and descending colon. The fluorescent dyes Fast blue (1%), Diamid±no yellow (1%) and Fluorogold (1%) were used for neu-

roanatomical tracing. A total of 10 microliters were injected into an individual organ using a 0.3-ml insulin syringe equipped with a 28-gauge needle (Becton-Dickinson); Fast blue into the ventral wall of the bladder at 3 - 4 injection sites; Diamidino yellow into the ventral wall of the descending colon at 3 - 4 sites; Fluorogold into the external urethral sphincter in one 10-p,l injection. Approximately 1 week later the animal was reanesthetized with pentobarbital (50 m g / k g , i.p.) and the abdominal cavity reopened. The major pelvic ganglia and the inferior mesenteric ganglia were removed and pinned

n

Fig. 1. Hematoxylin and eosin stained, 6-tzm-thick, transverse sections from the bladder body. A: diabetic. B: control. Note the large increase in both the diameter and wall thickness in the diabetic bladder compared to the control bladder. Bar = 2 mm.

279 out in a sylgard dish and immersed in saline for a short time to wash away superficial blood. Subsequently these tissues were exposed to fixative. The animal was then perfused transcardially with 250 ml of cold Krebs-Ringer followed by 300 ml of cold fixative (4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4). The following tissues were removed: (1) the spinal cord from its caudal end to T10 including the dorsal root ganglia from $2 to T10, (2) the bladder including the proximal urethra, (3) the L a - L 5 sympathetic chain ganglia. All tissues with the exception of the sympathetic chain were immersed overnight in buffer containing 25% sucrose prior to being sectioned on the cryostat. Tissues were sectioned at a thickness of 30 microns and thaw-mounted on gelatin-coated slides. Labelled neurons were examined under epifluorescent illumination and profiles drawn with the microscope drawing tube. To avoid measuring the same neuron more than once, only neurons with nuclei were drawn and only every other section was examined. The sympathetic chain was examined as a whole mount after being coverslipped with a non-fading mountant 17 under the same illumination. Cross sectional areas were measured using a digitizer board (Jandel Sigma Scan). Cell size distributions were prepared with Harvard Graphics and summarized as averages and standard deviations. Statistical comparisons were expressed using the two-tailed Student's t-test.

only a few labelled cells were found in the major pelvic ganglia. No labelled neurons were found in the spinal cord when tracers were injected into the bladder or the colon implying that there was no leakage from these structures to other tissues such as abdominal muscles or to the pelvic ganglia. The application of Fast blue to the central stump of one pelvic nerve labelled the intermediolateral preganglionic neurons of the ipsilateral sacral parasympathetic nucleus (SPN) as well as SCH and DRG neurons. Injections into the external urethral sphincter (Fluorogold) labelled the ventral horn motor neurons of the ventrolateral components of Onuf's nucleus. Table I presents the results and statistical analysis of measurements of the areas of labelled neurons for diabetic (D) and control (C) animals. The values in the 'pooled raw data' portion of the table are the result of combining all the data from equivalent experiments.. For the 'analysis of variance' portion of the table analysis was performed on the means of equivalent experiments. It can be seen that the average sizes of neurons in the MPG, DRG, IMG and SCH in diabetic animals were significantly larger than the corresponding neurons in control animals, for both the bladder and the colon. The percentage change between average neuronal areas of control and diabetic animals ranges between 13 and 68%. On the other hand the data for SPN neurons and Onuf neurons are not statistically different between diabetic and control animals. In addition, there was no difference between control and diabetic animals after measuring cell sizes in the superior cervical ganglion (data not shown).

RESULTS

Streptozotocin rats The bladders of STZ rats were distended and contained much larger volumes of urine than those of controls. When empty the diabetic bladder weighed more, was larger, and had a thicker wall than the normal bladder. Fig. 1 illustrates the large increase in diameter and in wall thickness of a diabetic bladder compared to a normal bladder. Bladder injections (Fast blue) labelled neurons in the major pelvic ganglia (MPG), the inferior mesenteric ganglia (IMG), the dorsal root ganglia (DRG) and the sympathetic chain ganglia (SCH). Colon injections (Diamidino yellow) labelled these same structures but TABLE I

Cell sizes in various tissues for diabetic (D) and control (C) animals Areas and standard deviations are in square microns.

Tissue Blad MPG Blad DRG Blad symp Chain Blad IMG Col DRG Col symp Chain Col IMG SPN ONUF

Analysis o f variance

Pooled raw Data (C) (D) (C) (D) (C) (D) (C) (D) (C) (D) (C) (D) (C) (D) (C) (D) (C) (D)

Area

S.D.

N

Area

S.D.

Cases

266.7 451.1 440.3 644.0 515.0 697.1 220.8 331.5 764.2 857.5 506.8 682.3 351.2 447.0 151.5 162.6 586.2 528.8

59.5 104.7 93.3 127.3 112.8 184.4 52.8 83.6 122.3 171.8 130.1 172.3 97.9 139.7 46.6 42.9 158.1 127.0

532 520 305 265 122 115 136 108 452 353 93 100 160 144 199 198 121 102

266.7 448.3 424.8 625.6 516.4 698.1 220.6 333.9 759.8 855.7 507.8 686.1 351.7 483.3 151.4 163.9 579.4 532.1

5.5 19.9 19.8 21.0 17.7 18.0 6.0 22.8 30.2 15.7 7.2 24.2 14.8 6.0 22.6 10.9 29.9 60.4

3 3 3 3 2 2 2 2 3 3 2 2 3 3 2 2 2 2

%Change

P

68.1

0.0~1

47.3

<0.0~1

35.2

0.01

51.4

0.02

12.6

0.~8

35.1

0.01

37.4

0.0001

8.3

0.556

- 8.2

0.426

280 350

300

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5200 O ~_1150 LU 0 100

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040

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20

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100 200 300 400 500 600 700 800 9 0 0 1 0 0 0 A R E A (sq microns)

A

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100 200 300 400 500 600 700 800 9 0 0 1 0 0 0 A R E A ( s q microns)

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150

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C

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100 200 300 400 500 600 700 800 9 0 0 1 0 0 0

0

100 200 300 400 500 600 700 800 90010001100

A R E A (sq microns)

A R E A ( s q microns)

Fig. 2. Size distributions of neurons labelled after dye was injected into the bladder body wall of diabetic (striped bars) and control (solid bars) animals. A: major pelvic ganglion. B: inferior meseneteric ganglion. C: dorsal root ganglia (data from L1, L2, L 6 and $1). D: sympathetic chain ganglia (data from L 3-L5). Note substantial shifts towards larger areas for diabetic animals.

Fig. 2 depicts the distributions of bladder-labelled neurons from diabetic and control animals. In all cases, the diabetic distribution is not only substantially shifted towards larger areas but the standard deviation of the diabetic distribution is quite a bit larger than that of the control distribution (see Table I). The sizes of neurons in the MPG and IMG are much smaller than the sizes of neurons from D R G s the sympathetic chain (Table I and Fig. 2). The average size of bladder MPG neurons is 2 6 7 / z m 2 and that of the bladder IMG neurons is similar. By comparison, bladder D R G neurons average 427 um 2 and bladder SCH neurons average 515 /zm 2. In addition, the sizes of bladder neurons in the MPG, D R G and the IMG are significantly smaller than those of the colon in these same tissues. For example, colon IMG neurons average 335/xm 2 and colon D R G neurons average 764 ~ m 2. There were no differences in the sizes of SCH neurons from these two organs. A comparison of D R G and IMG neurons from bladder and colon in control and diabetic animals was subjected to a two-way A N O V A analysis. The results reveal significant differences in average areas between bladder and colon as well as between control and diabetic animals when each factor was examined independently of the other (P values < 0.001). For sympathetic chain neurons this analysis resulted in a significant difference only between control and diabetic animals (P = 0.0001).

The relative frequency with which bladder and colon neurons were found in the D R G s examined is depicted in Fig. 3. It is notable that bladder neurons were found mainly in L 6 and S 1 with the majority in L6, whereas colon neurons were located mainly in S 1 and L 1 - L 2. Diuretic rats

The plasma glucose levels of these two rats were in the normal range (142, 166 m g / d l ) indicating that in spite of the high sucrose levels in the drinking water these animal were able to control their glucose levels and were not diabetic. On the other hand, the results 0.6 0.5 I-- 0 . 4 Z UJ (.,1 n- 0 . 3 UJ o. 0 . 2 0.1 0

L1

L2 DORSAL

L6 ROOT

$I

GANGLION

Fig. 3. Distribution of segmental locations of neurons labelled from the bladder and colon. Diabetic and control animals gave similar results and the c o m b i n e d data is s h o w n here. N o t e that bladderlabelled neurons are predominantly located in L 6 w h e r e a s colonlabelled neurons are mainly located in S 1 and L I - L 2.

281 TABLE II

Cell sizes in various tissues for diuretic (D) and control (C) animals Areas and standard deviations are in square microns.

Tissue Blad MPG Blad DRG Blad syrup Chain Blad IMG Col DRG Col syrup Chain Col IMG

Pooled raw data (C) (D) (C) (D) (C) (D) (C) (D) (C) (D) (C) (D) (C) (D)

Analysis of variance

Area

S.D.

N

Area

S.D.

Cases

266.7 429.7 427.3 693.4 515.0 600.0 220.8 335.2 764.2 759.6 506.8 508.5 334.7 362.7

80.0 89.6 87.9 163.3 112.8 152.4 52.8 99.8 122.3 157.7 130.1 131.8 97.2 93.6

532 286 530 232 122 121 136 105 452 123 93 70 58 100

266.3 428.8 424.8 693.8 516.4 600.7 220.6 335.9 759.6 759.4 507.8 514.6 351.7 362.7

5.5 2.6 19.8 7.8 17.7 6.6 6.0 4.9 30.6 20.3 7.2 8.6 14.8 11.6

3 2 3 2 2 2 2 2 3 2 2 2 3 2

of measurements in metabolic cages showed large increases in the average daily volumes of fluid intake and urine output; intake: 228, 192 ml, output: 213, 147 ml. Average fluid intake and urine output for control animals are 25.0+ 1.5 ml and 11.6+0.8 ml ( m e a n + S.E.M.) respectively. Furthermore, upon sacrifice, the bladders were grossly enlarged and weighed more than those of control animals (0.31, 0.38 g; average bladder weight of control animals: 0.16 + 0.01 g). The results of measurements of various cell sizes of these animals are presented in Table II. All neurons from regions that project to the urinary bladder including MPG, IMG, DRG, SCH are significantly larger than those of the control animals. On the other hand, neurons from these regions that innervate the colon show no significant changes in size when compared to the controls. In addition, except for the sympathetic chain, the increases in size are of the same order of magnitude as was found for the diabetic animals (Table I). DISCUSSION

In all cases where they made direct connections with the bladder or the colon, labelled neurons of diabetic animals were significantly larger than the corresponding neurons of control animals. These included neurons in the MPG, IMG, DRG and SCH. Changes in neuronal crossectional area imply even greater changes in the neuronal volume since the volume is related to area by the 3 / 2 power. Therefore, for example, the 61% increase in crossection observed for the bladder MPG neurons (Table I) implies an average volume increase of these neurons of 104%. On the other hand neurons which were involved with micturition but were separated from the endorgan by a postganglionic neu-

%Change

P

61.0

< 0.0001

63.3

< 0.0001

16.3

0.02

52.3

0.002

0.0

0.997

1.3

0.482

3.1

0.521

ron as in the case of the preganglionic neurons in the SPN, or which innervated the striated muscle of the external urethral sphincter (Onuf's nucleus), showed no significant difference in size between diabetic and control animals. Finally, neurons which are not at all concerned with the micturition process (i.e. the superior cervical ganglion, Table I) did not show any significant change in size. As shown previously 25'26 dorsal root ganglion neurons exhibited a somatotopic organization (Fig. 3); those innervating the bladder were preferentially located in L 6 whereas colon DRG neurons were predominantly found in S 1 and L1-L 2. The results from the diuretic animals (Table II) contrast sharply with those of the diabetic animals. Only those neurons innervating the bladder were hypertrophied whereas those that innervated the colon were unchanged from control animals. Diabetic animals exhibit polydipsia and polyuria but, in addition, they are also polyphagic and they hyperdefecate. By way of contrast, the diuretic animals displayed polydypsia and polyuria but did not hyperdefecate. In addition, both the diabetic and diuretic animals displayed bladder hypertrophy. It is also probable that the diabetic animals had hypertrophic colons since they defecated excessively, whereas the diuretic animals did not display excess defecation and therefore their colons were most likely normal. However, in neither case were the colons examined in this experiment. On the other hand, earlier reports w have demonstrated hypertrophy in other portions of the gastrointestinal tract (ileum and cecum) of SpragueDawley rats as well as severe neuropathy in the extrinsic nerves to those areas. In the present study these gross functional characteristics were reflected in the

282 measurements of neuronal sizes. Paralleling the hypertrophy of the bladder and colon in diabetic animals, the corresponding bladder and colon neurons were hypertrophic. By contrast in the diuretic animals only the bladder neurons were hypertrophic. Do the neurons innervating the bladder (or colon) become hypertrophic as a direct consequence of the diabetic condition or is neuronal hypertrophy a secondary phenomenon due to the stretching of the organ and its increased size? This type of organ hypertrophy is known as 'work hypertrophy' and has been discussed by Gabella 6. Since both bladder and neuronal hypertrophy occur in the case of the diuretic rat, it would appear that the diabetes is not the sole cause of hypertrophy in those animals. A question related to the hypertrophy of neurons innervating the smooth muscle of the bladder is: what changes occur in motor neurons innervating striated muscle when the muscle undergoes hypertrophy due to increased use? In a study of compensatory hypertrophy 4 of the rat plantaris muscle investigators reported a 32% decrease in motor neuron volume corresponding to a 65% increase in muscle weight. This apparently contradictory result most likely reflects a transformation of the plantaris muscle cells from fast to slow type with a consequent change in the motor neurons (D. Finkelstein, private communication). The hypertrophy of the diabetic or diuretic bladder and that of the neurons innervating it raises the question of the mechanism(s) through which this phenomenon occurs. One response is that since the target increases in size and weight there is more tissue to innervate and therefore the relevant neurons increase their size due to the increased demand. In fact increases in the levels of cholinergic and adrenergic markers ~° and upregulation of muscarinic receptor density 9 for the diabetic bladder have been reported. However this answer does not provide an explanation of the nature of the message(s) from the target to the neuron, which might be a chemical such as a growth factor. Such a situation has been suggested in the case of the obstructed rat bladder and in fact Steers et al. found a large increase in the level of nerve growth factor (NGF) in the bladder preceding the hypertrophy of neurons of the major pelvic ganglia 24. Numerous studies have determined that, in the periphery, N G F is made in target tissues and is transported by retrograde axoplasmic flow to the neurons innervating these tissues where it is used to support vital metabolic processes (for review see Snider and Johnson21). In the case of the diabetic animal there have been several reports suggesting that the level of nerve growth factor decreases in many of the tissues

examined TM. Some tissues (e.g. vas deferens, prostate) showed an increase in N G F content, some tissue (e.g. heart atrium, ileum) showed no change, and in some tissues the levels increased early after diabetes and decreased at later times (e.g. heart ventricle). There have been no reports of measurements of NGF levels in the diabetic bladder. In addition, Schmidt et al. have shown that in the diabetic rat, the retrograde transport of N G F from the target tissue to the innervating neurons may be compromised, resulting in a lower amount of transported N G F 2°. Thus, in the case of the diabetic bladder not only might the tissue level of N G F be affected but also its transport. Other examinations of the axonal flow process in diabetic animals have reported reduced speeds 1'13 of varying amounts dependent upon which particular axonal component was being studied. Therefore other neurotrophic factors (presently unknown, Barde 2) or a reduction, due to compromised axonal flow, of a normally present trophic factor may be responsible for the observed hypertrophy in the groups of neurons innervating the bladder. In summary, the STZ-diabetic rat exhibits hypertrophy of the urinary bladder (and most likely the colon) along with hypertrophy of those sensory and postganglionic neurons that make direct contact with these organs. Neurotrophic substances which might be implicated in this process are presently unknown. Acknowledgements. This work was supported by funds from the

veterans administration and the Department of Neurological Surgery, University of Pittsburgh Medical School(Copeland Fund). We would like to thank J. Johnston and A. Nguyen for technical assistance.

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