Author Institution: Department of Physiology, Ohio State University, College of Medicine, Columbus, Ohio 43210The pattern of development of phenylhydrazine-induced rat reticulocytosis was studied over a period of nine days. Intraperitoneal injections of phenylhydrazine (4 mg/100 gm) every other day caused a fall in hematocrit which leveled off at 60% of normal by the fifth day. Increased erythropoiesis was indicated by a three-fold increase in the number of circulating reituclocytes after the first three injections. The immediate response was the release of stored mature reticulocytes from the bone marrow. As the anemia progressed, more and more young reticulocytes appeared until 70 to 85% of the red cells in the peripheral circulation were reticulocytes and 20% of these were juvenile forms

Flanagan, J. Patrick

Lessler, Milton A.

The Ohio Journal of Science

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CONTROLLED PHENYLHYDRAZINE-INDUCEDRETICULOCYTOSIS IN THE RAT1-2J. PATRICK FLANAGAN3 AND MILTON A. LESSLERDepartment of Physiology, Ohio State University, College of Medicine, Columbus, Ohio 43210ABSTRACTThe pattern of development of phenylhydrazine-induced rat reticulocytosis wasstudied over a period of nine days. Intraperitoneal injections of phenylhydrazine (4mg/100 gm) every other day caused a fall in hematocrit which leveled off at 60% of normalby the fifth day. Increased erythropoiesis was indicated by a three-fold increase in thenumber of circulating reituclocytes after the first three injections. The immediate re-sponse was the release of stored mature reticulocytes from the bone marrow. As theanemia progressed, more and more young reticulocytes appeared until 70 to 85% of thered cells in the peripheral circulation were reticulocytes and 20% of these were juvenileforms.INTRODUCTIONAdministration of phenylhydrazine to experimental animals causes an increasein erythropoiesis, as evidenced by the release of reticulocytes into the circulatingblood. Phenylhydrazine is an oxidant drug which destroys red cells by its effecton enzymes involved in energy metabolism (Jandl et al., 1960). It results in thedenaturation of red cell hemoglobin in vitro or in vivo with little evidence of toxicityto other tissues, if the dosage is carefully adjusted (Cruz, 1941; Rifkind andDanon, 1965). The drug primarily damages mature erythrocytes inducing anerythropenia which, in turn, leads to an accelerated erythropoiesis (Smith andMcKinley, 1968). Controlled bleeding of animals also results in acceleratederythropoiesis, but the reticulocytosis evoked by phenylhydrazine is more rapidand is more easily controlled for experimental purposes (Jacobson et al., 1948).In order to investigate the cytological and biochemical changes that occur duringreticulocyte maturation, it was necessary to obtain rats with large numbers ofcirculating reticulocytes. Studies of phenylhydrazine anemia enabled us toproduce rats with predictable percentages of reticulocytes in their blood.Reticulocytes appear in the circulation as non-nucleated cells that completetheir hemoglobin synthesis and differentiate into erythrocytes in approximatelytwo days (Lowenstein, 1959; Plum, 1949). When supravitally stained withdilute solutions of Brilliant Cresyl Blue (Heath and Daland, 1931) or NewMethylene Blue (Brecher, 1949), reticulocytes show characteristic granules or areticular network. Transitional forms of reticulum have been described andseveral investigators have classified reticulocytes by the amount, form, and dis-tribution of stained reticulum (Heilmeyer, 1932; Jandl, 1960; Seip, 1953). Jensenet al. (1965) recently demonstrated that the basophilic stippling of reticulocytesis due to aggregation of the ribosomes present in the maturing cells. As reticulo-cytes mature into erythrocytes, their ribosomes gradually disappear and less andless stainable reticulum is evident. There is general agreement that reticulocytematurity is inversely proportional to the amount of stained reticulum, and thatthe course of a reticulocytosis can be analyzed by the types of reticulocytes thatappear in stained blood smears. Reinvestigation of phenylhydrazine-inducedxThis investigation was supported by the VanWert United Health Fund, a USPHS GeneralResearch Support grant, and Grant AM 09326 from The National Institute of Arthritis andMetabolic Diseases.2Manuscript received April 3, 1968.3J. P. Flanagan, a medical student, was awarded a Roessler Foundation Scholarship toassist him in this work.THE OHIO JOURNAL OF SCIENCE 70(5): 300, September 1970.No. 5 PHENYLHYDRAZINE-INDUCED RETICULOCYTOSIS 301anemia enabled us to obtain rats with high percentages of young or maturereticulocytes in their circulating blood, and to study the course of reticulocyterelease and maturation (Lessler et al., 1967).METHODS AND MATERIALSReticulocytosis was induced in mature male Wistar rats by interperitonealinjection of phenylhydrazine hydrochloride (4 mg/100 gm of rat) on days 1, 3, 5, 7,of a nine-day experimental period. The initial weight of the rats ranged from300 to 490 grams, and the final weights (day 9) from 280 to 430 grams. Theanimals were watered and fed Purina lab-chow without added supplements of iron.The rats were weighed, and blood samples were obtained from the tail veinjust before each phenylhydrazine injection and also on the 9th day, 48 hoursafter the final injection. Duplicate microhematocrits and blood smears weremade from each blood sample. Supra vital staining of reticulocytes was done bymixing one drop of blood with one drop of 0.5% New Methylene Blue in 0.65%potassium oxalate and incubating for 10 minutes in a moist chamber at roomtemperature before smearing (Brecher, 1949). The air-dried New-Methylene-Blue-stained smear was counterstained with Wright's stain, and 1000 red cellswere counted on each of two duplicate slides with an oil-immersion objective.Reitculocytes were counted as young (group I, II) or mature (group III, IV)cells, according to the amount and distribution of stained reticulum. Based onthe classification of Heilmeyer and Westhauser (1932) and of Seip (1953), group Ireticulocytes have a dense coherent reticulum filling the greater part of the cell.Group II reticulocytes have an extended reticulum, which is looser than that ingroup I because of occasional breaks in the network. Group III reticulocyteshave some small bits of reticulum, and group IV cells have only a few bluegranules and are essentially erythrocytes.Counts were made by two independent investigators over a period of twoyears on a series of 20 rats experimentally treated in groups of three to four withconsistent results. These data were averaged, and the standard deviations(Table I) show a relatively small spread, except for the day-seven and day-ninerats, which were severely anemic. One control (untreated) rat was counted witheach group; these animals showed little variation from the 2% reticulocytesobserved in colony rats whose blood was studied before any treatment had beengiven.RESULTSThe pattern of development of the phenylhydrazine-induced anemia wasevident from the fall in hematocrit from a value of 49, in untreated rats, to 30(Table 1). Hematocrit values fell 36% after 2 injections (4 days) as the percentageTABLE 1Average values of phenylhydrazine induced reticulocytosis in rats1Day13579Hematocrit -49±1.8238±2.531±2.530±3.929±4.8Total2.2± 07.4=*= 022.5± 552.0±1270.1=*= 9.7.8.6.6.1Group0.0±00.7±02.2±16.5±46.0±4Percent reticulocytesI.0.5.6.3.4Group II0.2±0.50.9±0.73.9±2.111.1±6.312.9±7.8Group III0.4±0.42.2±1.46.8±2.616.2±6.722.9±4.4Group1.6="=3.6=*=9.2±18.2±28.3±IV0.61.22.89.59.51Initial weights ranged from 300 to 490 grams.2=t standard deviation of the mean.302 J. PATRICK FLANAGAN AND MILTON A. LESSLER Vol. 70of reticulocytes increased to approximately 30% of the total circulating red-cellpopulation (fig. 1). The decrease in hematocrit tended to level off at about 30,by the fifth day, but the percentage of reticulocytes, which rose from 2 to 20%in five days, more than doubled by the seventh day (after 3 injections). On theninth day (after 4 injections) the hematocrit was 29, but 70 to 80% of the redcells in the peripheral blood were reticulocytes. Juvenile reticulocytes (groupI, II) increased with each injection until they made up approximately 20% of thered-cell population in the anemic rats on days seven and nine (Table 1).During the course of the developing anemia, the average weight of the animalstended to fall, with the greatest decrease seen after the second injection (fifth day).There was an inverse relationship between percent of reticulocytes and weight loss.The greatest weight loss was apparent on the fifth and seventh days and occurredafter the animals had developed a severe reticulocytosis (Fig. 1). The firstHct.5 550454 03 53 025% Wt.Retic. Gm.4 404 2 04 0 033 03 6 0340320FIGURE 1. Relationship of hematocrit and body weight to percent reticulocytosis in Wistarrats injected with phenylhydrazine (40 mg/kg) on days 1, 3, 5, and 7.No. 5 PHENYLHYDRAZINE-INDUCED RETICULOCYTOSIS 303phenylhydrazine injection resulted in an increase in reticulocytes from 2 to 7%.This was due mainly to the release of mature reticulocytes. Successive injectionsof phenylhydrazine resulted in an increase in the percentage of young reticulocytesin the peripheral blood until they made up approximately 33% of the circulatingreticulocyte population after the third and fourth injections (Table 1).DISCUSSIONUnder normal laboratory conditions, healthy Wistar rats have 2 to 3%reticulocytes in their peripheral blood and these are almost exclusively matureforms (group III, IV). Reticulocytes in the circulation mature into erythrocytesin approximately two days. A single injection of phenylhydrazine destroysmany of the red cells as well as some of the mature reticulocytes (Jandl et al.,1960; Cruz, 1941). The destruction of red cells causes an anemia which stimulatesthe release of the mature reticulocytes normally stored in the bone marrow.Although erythropoiesis was increased, as indicated by the small rise in immatureforms, the anemia caused by a single injection of phenylhydrazine was not severeenough for the early release of large numbers of young reticulocytes. Each succes-sive injection of phenylhydrazine, however, challenged the animals to increaseerythropoiesis further in order to replace the destroyed red cells. This they did,as indicated by the increase in the number of young reticulocytes released afterthe second, third, and fourth injections.There is a gradient of increasing susceptibility of red cells to the denaturativeeffects of phenylhydrazine and related compounds which is directly proportionalto cell age (Cruz, 1941). Thus, mature erythrocytes are more susceptible thanare reticulocytes or the reticulocyte precursor cells. Injection of phenylhydrazineevery other day resulted in a progressive anemia because of the selective destruc-tion of mature red cells which resulted in an erythropoietic stimulation of the bonemarrow. After each injection the bone marrow had two days to increase erythro-poiesis and release reticulocytes. The reticulocytes in the circulation also maturedinto erythrocytes during that period. Thus, it was possible to destroy additionalerythrocytes and the reticulocytes that had matured into erythrocytes after eachof the subsequent injections with further stimulation of bone marrow erythro-poiesis. Controlled reticulocytosis by alternate-day injections of phenylhydrazineworks best with mature male rats (300-400 gm). Similar injections given toyoung rats (125-150 gm) causes a severe anemia and many of the animals dieafter one or two injections. Mature male rats survive all four injections and havelarger numbers of circulating reticulocytes (70 to 85%) than can be obtained bysequential bleeding (Jacobson et al., 1948). Although reticulocytosis can beinduced in animals by other doses of phenylhydrazine, alternate day injectionsof 4 mg/100 gm make it possible to routinely prepare anemic rats with 70% ormore of their circulating red cells as reticulocytes.LITERATURE CITEDBrecher, G. 1949. New Methylene Blue as a reticulocyte stain. Am. J. Clin. Path. 19:895-896.Cruz, W. O. 1941. Acetylphenylhydrazine anemia. I. The mechanism of erythrocytedestruction and regeneration. Am. J. Med. Sci. 202: 781-789.Heath, C, and G. Daland. 1931. Staining of reitculocytes with Brilliant Cresyl Blue. Arch.Int. Med. 48: 133-145.Heilmeyer, L., and R. Westhauser. 1932. Studies on the ripening of surviving reticulocytesin vitro and their significance in the evaluation of daily hemoglobin production in vivo.Zitschr. Klin. Med. 121: 361-379.Jacobson, L. O., C. K. Marks, E. O. Gaston, E. L. Simmons, and M. H. Block. 1948. Studieson radiosensitivity of cells. Science 107: 248-250.Jandl, J. H., L. K. Engle, and D. W. Allen. 1960. Oxidative hemolysis and precipitation ofhemoglobin. I. Heinz body anemias as an acceleration of red cell aging. J. Clin. Invest.39: 1818-1836.304 J. PATRICK FLANAGAN AND MILTON A. LESSLER Vol. 70Jensen, W. N., G. D. Moreno, and M. C. Bessis. 1965. An electron microscope descriptionof basophilic stippling in red cells. Blood 25: 933-943.Lessler, M. A., J. P. Flanagan, and D. L. Beerman. 1967. Reticulocyte respiratory activityas an index of cell maturity. Fed. Proc. 26: 770.Lowenstein, L. H. 1959. The mammalian reticulocyte. Int. Rev. Cytol. 8: 135-174.Plum, C. M. 1949. On the reticulocytes, their occurance and development. Acta Haematol.2: 317-348.Rifkind, R. A. and D. Danon. 1965. Heinz Body Anemia—An ultrastructural study. I.Heinz body formation. Blood 25: 885-896.Seip, M. 1953. Reticulocyte Studies. Acta Med. Scandinav. Suppl. 282: 9-164.Smith, L. H., and T. W. McKinley, Jr. 1968. Protection of mice against radiation injury byphenylhydrazine. Proc. Soc. Exp. Biol. Med. 129: 702-705.

Controlled Phenylhydrazine-Induced Reticulocytosis in the Rat

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Controlled Phenylhydrazine-Induced Reticulocytosis in the Rat

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