E. A, Shanahan
Under the combined Science/Medicine Scheme, under-graduates have been offered the opportunity of obtaining the degree of Bachelor of Science by engaging in an addi- tional year's work, approved by the Faculty of Science. For many years this scheme has been limited to two alternatives—com- pletion of two years of an approved Science course before transference to M.B. B.S. or interruption of the Medical course after Division I or II for further study in related medical subjects and pre-requisite science subjects. In recent years the scheme has been extended to include the so-called
"investigational method". The latter con- sists of the engagement of the student in investigational work in departments such as Physiology, Anatomy, Pharmacology, Bio- chemistry, Bacteriology, Experimental Medicine and Pathology, for one calendar year. The actual form of the course is left mainly to the discretion of the Professor of the Department, who may also recommend the study of additional subjects. On com- pletion of the year a report of the investi- gational work must be submitted and examination may be necessary.
Last year, having completed Division II and the interim term of Division III, I interrupted my Medical course in order to undertake a year of investigational work in Pathology under the regulations of the above scheme. On the recommendation of Professor King I also enrolled for Chem-
istry II and Science German. The Thomas and Elizabeth Ross Scholarship is open to under-graduates wishing to pursue a com- bined Science/Medicine course and in 1959 a living allowance of £500 was provided with an additional sum for books and appa- ratus. Commonwealth Scholarships will extend their scholarships for the year, pay- ing all compulsory fees. Hence from the financial, aspect the additional year is in no way expensive.
The actual Pathology course consisted of the Science Pathology practical course which includes Experimental Pathology, Histo-Pathology and Chemical Pathology; a six-week course in Microscopy and the study of the post-graduate series of patho- logical histology sections; attendance at and performance of post-mortems, and instruction in various techniques such as frozen sectioning, tumour transplantation and bone-marrow biopsy. Most of the year was spent in investigational work, the aim of which was directed more toward the learning of techniques and investigational methods than to original research.
At this stage it may be of interest to elaborate on the Science Pathology practical course. Experimental Pathology, as the name implies, is concerned with the use of experimental animals as an aid to research.
For example, the original Goldblatt experi- ment of the production of hypertension following ligation of the renal artery was
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repeated using rats and chemical carcino- genesis using benz-pyrine was studied in mice. Histo-pathology involved the study of and practical work in paraffin and celloidin blocking, cutting of sections and staining techniques. The Chemical Patho- logy section is as yet in its infancy but deals with such things as absorption histo- Spectroscopy and auto-radiography. Dur- ing the year visits were arranged to the tissue-culture laboratories of C.S.I.R.O.
and the smear-cytology department of the Royal Women's Hospital.
The investigational work in which I was engaged consisted mainly of micro-dissec- tion of abnormal and normal nephrons, although the screening of substances for carcinogenic activity, in particular extracts Of starch heated to 400°C and lard prepara- tions, was carried on throughout the year.
The first six weeks of the year were spent on the micro-dissection of normal human nephrons. The data obtained from this dissection was used to establish a library of the dimensions of normal nephrons from humans of all ages. The remainder of the Year was devoted to the investigation of the changes occuring in individual nephrons and the kidney as a whole following the surgical removal of 75 per cent. of renal tissue.
The hypertrophy of renal tissue follow- ing the removal of portion of the kidney either by surgery or some destructive patho- logical process created much interest in the first 30 years of this century. Early work- ers found that the increase in size of the remaining kidney was due chiefly to an increase in the volume of the cortex, as a result of hypertrophy and a slight hyper- plasia of the glomeruli and convoluted tubules. The lesser increase in the volume of the medulla was due to dilatation, there being no increase in the number or size of the constituent cells. In 1924, Oliver (who is regarded as the father of nephron micro-dissection), investigated by histo- logical methods the morphological changes in the remaining kidney following unilateral nephrectomy. He found, by measurement of cross-sectional areas, that the glomeruli and convoluted tubules were definitely en- larged but there was no evidence of an Increase in their number, while the ascend- ing limbs of Henle's loops and the collect-
ing ducts showed only a slight increase in size. He concluded that the hypertrophy of the nephron is most marked in the proximal convoluted tubule and that the weight of the hypertrophied kidney could not be used as an index of the amount of functional tissue present. Addis in con- junction with Oliver, investigated the change in renal function in the same experi- mental animals and found it to be appar- ently unaffected by unilateral nephrectomy.
In the experiment I conducted two series of rabbits, eighteen in all, were used. The first series were semi-loops and the second ordinary domestic rabbits. Each series contained both male and female rabbits, all being fully-grown, ranging in weight from 1774 Gm. to 3008 Gm. All were fed normal diets. Hemi-nephrectomy was per- formed and followed two to six weeks later by total nephrectomy on the opposite side.
The animals were administered penicillin pre- and post-operatively. The remaining renal tissue was removed at post-mortem, the rabbits being sacrificed at intervals of one month. The hemi-kidney removed at the first operation and the kidneys of those animals which died during operation were used as controls. All kidneys and portions of kidneys were weighed and their dimen- sions recorded after 48 hours fixation in 10 per cent. formol saline. Haemotoxylin and eosin sections of all kidneys and organs were made. Frozen sections of both normal and hypertrophied kidneys were stained with iron haemotoxylin and Van Giesons stain following the methods of Oliver, these sections being used for the measurement of nephron diameters. In each case 20 measurements of the greatest diameter of the glomerulus and capillary tuft were taken. Three sets of twenty measurements were made on the collecting tubules, proximal convoluted tubules and distal convoluted tubules. These were the diameter from basement membrane to basement membrane, the height of the epithelium and the diameter of the lumen of the tubule. Only those cross-sections which were practically circular in outline were selected for measurement.
Blood urea estimations were carried out before operation, after hemi-nephrectomy and after total nephrectomy where possible, but time did not allow extensive investiga- tion of blood and urine chemistry.
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32 SPECULUM
The three-dimensional morphology of individual nephrons was studied by micro dissection employing the technique of Oliver. After fixation in 10 per cent.
formol saline for at least one week, the kidney was cut into slices 3-4 mm. in thick- ness and then macerated in concentrated HC 1 at a temperature of 22°C. The time of maceration varied from 35-106 hours depending upon the external temperature and the degree of fibrosis of the kidney tissue. The macerated tissue was dissected under water using a stereoscopic micro- scope at a magnification of 50. The dis- sected material was then mounted in a drop of water and photographed with a Leica camera using 35 mm. Ilford micro-negative pan film
The mortality resulting from operation and post-operative complications was exceptionally high and of the eighteen rabbits used only six survived a sufficient length of time to give significant results.
The extremely high incidence of post- operative infection was apparently unavoid- able under the experimental conditions which prevailed. The utmost care was taken to maintain a surgically clean although not sterile field. Chloramphenicol was found to be most effective in combating any renal tract infections which developed later.
It was obvious from the early stages of the experiment that the weight of the kidney removed at post-mortem was not an accur- ate index of the amount of functional tissue as, apart from the alteration in the normal volume or size relationship of the proximal convoluted tubule to the remainder of the nephron, fibrous tissue contributed a large and variable proportion of the total weight.
dilatation over the six-month period, the distal convoluted tubules and collecting tubules showed slight hypertrophy and much dilatation and interstitial fibrosis varied in amount with time, being most prominent in the six month kidney. Mitotic figures were not seen but nuclear condensa- tions (query nucleoli) were numerous in the one month kidney. The tissues of the rabbit sacrificed at six months were of particular interest in that deposition of calcium had occurred in the basement membranes of the glomeruli and tubules, some presenting the appearance of psammoma bodies, and also in the media and intima of the aortic ring and ascending aorta where the inorganic material had excited a foreign-body giant- cell reaction. All organs showed varying degrees of arteriolar sclerosis, suggestive of an elevation in blood pressure.
Photomicrograph of cortex showing a glomeru- lus with dilated capsular space, fibrosis and
hypertrophic and dilated tubules.
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All organs were examined macroscopic- ally and microscopically. The kidney remnants removed at post-mortem were enlarged to two or three times their esti- mated original size. Calculi, probably of secondary origin were a constant finding.
Microscopically the capsules were thick- ened, the cortices consisted of alternating areas of hypertrophic and normal proximal convoluted tubules, the glomeruli were normal in number but showed progressive
The normal range of blood urea levels in the rabbit are comparable with those of man, i.e., 15-30 mgm. per cent. Normal rabbits, used as controls, were found to have levels between 17 and 19 mgm. per cent. On the whole the blood urea levels remained within normal limits but each operation was followed by a slight rise of approximately 8 mgm. per cent. Only when infection and renal failure supervened
did the blood urea rise above the upper limit of normality.
A practically constant percentage in- crease (approximately 46 per cent.) in the cross-sectional diameter of the proximal convoluted tubule agreed remarkably with the figures of Oliver (approximately 45 per cent.) and the attainment of this value at one month suggested that most of the increase in size occurs in this period.
Although more variable and of lesser degree, there was an increase in size of other portions of the nephron, due to both hypertrophy and dilatation. Hyperplastic changes, indicated by increased numbers of mitotic figures, were not observed but this does not exclude the possibility of such a process contributing to the increase in nephron size, as observations in rats have shown that mitotic figures increase during the first week or so following the removal of renal tissue and having attained a maxi- mum rapidly decrease in number. It is probable that a similar phenomenon occurs in rabbits.
Micro-dissection studies revealed that the end results of the processes of hypertrophy, manifested as an increase in tubular length, diameter and the degree of convolution were definitely predominant in the proxi- mal convoluted tubule; the intermediary stages of fine convolution, festooning and diverticulum formation being evidenced throughout but predominating in the early stages, i.e., the first three months. The glomerular increase in size was due mainly to dilatation, however the capillary tuft showed an increase in diameter suggestive of increased blood flow through the kidney.
According to Oliver the formation of a neck (a constant finding in this series) is due to the portion of tubule contiguous with the glomerulus remaining undilated, thus form- ing a long narrow stretch extending to the distended portion of the proximal convo- luted tubule. Dilatation of the capsular space and the nephron as a whole increased gradually with time and in the case of tubular portions, dilatation appeared to be ascending, the collecting and distal convo- luted tubules being the first to exhibit this change. Dilatation predominated over hypertrophic changes in the six month kidney. This particular kidney presented both histological and micro-dissection pic-
tures very similar to, but less advanced than, those of chronic nephritis. The terminal one-third of the proximal convoluted tubules showed the most obvious changes of convolution compared with their normal straight structure. Many hypertrophic changes were masked by those due to fibrosis which was presumably of infective or ischaemic origin. Rupture of the tubular basement membranes from either ischaemia, mechanical injury during surgery or infection and the subsequent fibrosis and disordered regeneration which may follow could have been responsible for the forma- tion of the large tubular and glomerular cysts noted in the four and six month kid- neys. As the normal kidneys from these animals did not contain any of these struc- tures it is unlikely that they have resulted from any developmental anomaly.
High power view of glomerulus with "neck"
Apart from being statistically insignifi- cant on the basis of number, the dimensions of the nephron determined from micro dissected material are of doubtful reliabil- ity, as the tubules were observed to swell when placed in water, the degree of swell- ing increasing rapidly with time.
Of particular interest was the finding of calcification of Bowman's capsule, the tubular basement membrane and the media of the ascending thoracic aorta in the rabbit sacrificed at six months. The rabbit shows a peculiar tendency to undergo heterotopic calcification and ossification in the kidney
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