British Journal ofhlaematotogy. 1977, 35,

233.

Biphasic Thrombopoietic Response to Severe Hypobaric Hypoxia CARLW. JACKSON AND CAROL C. EDWARDS

Laboratory of Hematology, St Jude Children’s Research Hospital, Memphis, Tennessee, U.S.A. (Received

10 M a y

1976; acceptedfor publication 28 July 1976)

SUMMARY. Thrombopoiesis has been studied during and after an 11 d exposure to discontinuous hypobaric hypoxia. Exposure of rats to 0.4 atmospheres for 16-17 h daily initially caused an increase in platelet count which reached a peak of 1.5 times baseline on days 4 and 5. This thrombocytosis was followed by a decrease in platelets to a nadir of 5 ~ 6 0 %of baseline on days 12 and 13. That thrombocytosis results from increased platelet production is supported by increased [35 Slsulphate incorporation into platelets and increased megakaryocyte size and turnover. The thrombocytopenia with continued hypoxia seems to result from decreased platelet production since Cr-platelet survival was normal while megakaryocyte concentration was decreased to one-half that of untreated controls. These observations suggest that differentiation of precursors into niegakaryocytes was decreased during the thrombocytopenic period, although the fewer remaining megakaryocytes appeared stimulated because of their larger size and increased [3 Hlthymidine labelling. Thus, hypobaric hypoxia had a biphasic effect on thrombopoiesis with increased platelet production in the first few days of exposure followed by subnormal production. The erythropoietic response to hypoxia is well known. However, studies of the effects of hypoxia on thrombopoiesis are few and the available results inconsistent. In a report by Kingma & De Langen (1955), five rabbits exposed daily in hyobaric chambers to a pressure of 310 mmHg had increased platelet counts on day 4. In contrast, in experiments described by Birks et a1 (1975) thrombocytopenia developed in mice after 5-9 d of continuous exposure to 0.5 atmospheres of pressure in hypobaric chambers. Other investigators have reported no significant change in platelet count in rats (de Gabriele & Penington, 1967) or Rhesus monkeys (Garvey et al, 1969) chronically exposed to one-half atmosphere. Our interest in studying platelet production during exposure to hypobaric hypoxia was prompted by observations that acute anaemia stimulated megakaryocytopoiesis and increased the platelet count (Jackson et al, 1974). We report here studies on platelets and megakaryocytopoiesis in rats exposed to hypobaric hypoxia. We have used approximately the same conditions described by Camiscoli & Gordon (1970) for preparation of polycythaemic animals used in erythropoietin assays in which the erythroid response has been studied in detail. Correspondence: Dr Carl W. Jackson, StJude Children’sResearch Hospital, 332 N. Lauderdale, Memphis, Tenn. 36101, U.S.A.

23 3

234

Carl W.Jackson and Carol C. Edwards MATERIALS AND METHODS

Male Long-Evans rats weighing 278-377 g were obtained from Blue Spruce Farms, Inc., Altamont, N.Y. Platelets were counted by the phase contrast method (Brecher & Cronkite, 1950).Packed cell volumes were determined by a micromethod. Collection of blood samples. Blood was obtained by puncturing a vaseline-coated tail vein with a 25-gauge needle. Blood for platelet counts was obtained using the Unopette system. Blood for PCV determination was collected in heparinized 75 mrn capillary tubes. ExposcIre to hypobaric hypoxia. Rats were exposed to 0.4 atmospheres of hypobaric hypoxia in 55-gallon cylindrical drums which were fitted with reinforced plastic tops. The drums were connected to a rotary evacuation pump and were equipped with a pressure gauge manometer and an intake value that opened to normal atmospheric pressure to allow a flow-through of air. Rats were exposed to hypobaric pressure for 1 6 1 7h daily beginning about 16.00hours. Food and water were provided at all times. The rats were removed from the chambers at about 9 . 0 0 hours for cage changes, feeding and watering.

Blood Volume Estimation Blood volume was estimated using the 5gFe-labelled erythrocyte dilution technique. Rats weighing approximately 500 g which served as erythrocyte donors were given a single injection of 20 PCi of [5gFe]ferriccitrate intraperitoneally. Eleven days later blood was collected from the aorta into 3.8% sodium citrate. The blood was diluted with saline (I part saline: 2 parts blood), the blood cells separated by centrifugation and resuspended with an equal volume of saline. Three millilitres of blood were removed from a jugular vein of recipient rats and replaced with 3 ml of the "Fe-labelled erythrocyte suspension. Samples for determination of 5gFe-radioactivity were collected with the Unopette system for platelet counting using a 20~1 capillary pipette. The Unopette reservoir was assayed for radioactivity in a Packard gamma scintillation spectrometer, Blood volume was expressed as a per cent of body weight. Determination of Platelet Survival Platelet survival was estimated using "Cr (Aster, 1967).Platelets were separated by differential centrifugation from whole blood collected in acid citrate from the aorta of donor rats. The platelets were resuspended in Hanks' balanced salt solution without calcium and with one-half the magnesium ion concentration and incubated with NaZ5'CrO, at room temperature. After incubation the platelets were pelleted and washed twice with rat plasma diluted I :I with the Hanks' balanced salt solution described above and resuspended in the plasmaHanks' solution for transfusion. Chromium bound to contaminating erythrocytes accounted for 1.4-4.2% of the radioactivity in the labelled platelet suspensions from untreated rats and 5.4-5.5% in those from rats exposed to hypoxia. Blood samples ( 2 0 ~ 1 from ) the recipients were obtained using platelet Unopettes at 1-2, 24,48, 72 and 92 or 96 h. The radioactivity was determined in a Packard gamma scintillation spectrometer. To construct platelet survival curves, the radioactivity of the later time periods was expressed as a per cent of the initial count (I -2 h). The proportion of the platelet radioactivity injected which was circulating at 1-2 h was also calculated.

'

Thrombopoietic Response to Hypoxia

23 5

’’

Platelet production rate as measured by [’ S]strlphate incorporation into platelets. [ Slsulphate was given in a dosage of I pCi/g body weight 24 h prior to killing. The platelets were separated from blood collected in acid citrate, washed three times with Hanks’ balanced salt solution described above, solubilized in NCS tissue solubilizer and placed in scintillation fluid, The radioactivity was determined in a liquid scintillation spectrometer. [’ Slsulphate incorporation into platelets is expressed as a per cent of the [35S]sulphate injected. Tritiated thymidine labelling index of bone marrow megakaryocytes. Rats were given [’HIthymidine ( I pCi/g body weight )intravenously 24 h prior to killing. At killing, tibia1 bone marrow smears were prepared and fixed 2-30 niin in absolute methanol. Autoradiograms were prepared by dipping the smears in Kodak NTB-2 emulsion, exposing in light-tight boxes at 4°C for 4 weeks, developing in Kodak D-19 developer and staining with Giemsa. The number of labelled and unlabelled megakaryocytes along one lateral edge of the smear was determined and the labelled cells were expressed as a per cent of total megakaryocytes. The megakaryocytes were also classified morphologically as immature, mature or naked nuclei. Borrc rnarrow rrregakaryocytc~corrcmtratiorr. The average number of megakaryocytes per high power field was determined on 4-6 pni sections of femoral marrow that was fixed in 10% formalin, decalcified and stained with haeniatoxylin and eosin. One entire longitudinal section was evaluated for each animal. Megakaryocyte diametcr. Megakaryocyte diameter was derived using an eyepiece micrometer. The diameter was expressed as the square root of the product of two measurements made at right angles. Twenty-seven megakaryocytes adjacent to the feather edge of each smear were measured at a magnification of x 1250. Fifty megakaryocytes in feinoral marrow tissue sections from each of six untreated controls and six rats exposed to hypoxia for 11, 14 or 17 d were measured at a magnification of x 1000 for correction of inegakaryocyte concentrations of those time periods for increased megakaryocyte size. Means were aiialysed using the Studcnt’s t-test. The niegakaryocyte size distributions were aiialysed using a two-sample rank test.

RESULTS Platelet Cotitit arid PCV Platelet counts and PCVs during exposure to 1 1 d of discontinuous hypoxia (0.4 atmospheres) are shown in Fig I . Platelet counts increased beginning on day 2 and peaked at 1.5 times baseline on days 4-5. Thereafter the platelet count declined, reaching baseline values 011 day 7. However, instead of levelling out at baseline, the platelet count declined to a nadir of 50-60% on days 12 and 13. After discontinuation of the hypobaric hypoxia, the platelet count gradually increased back to baseline by day 18. The packed cell volume had increased froin 46 to 60% by day 4, peaked at 70% on day 1 1 when hypoxia was discontinued, then slowly declined to 62% by day 18. There was an average body weight loss of 14.5% during the first 2 d with that weight retained for the duration of exposure to hypoxia. Blood Volume Changes As shown in Fig 2, the relative blood volume of hypoxic rats was significantly increased on

Carl W .Jackson and Carol C . Edwards

236

t

Hypoboric hypoxio (0.4 otm)

0 ~ '4 '~

'

I

'

5

'

' ' '

I

10

15

HHCICIHHHHHI-4

40t

' '

Tlme (d)

Time (d)

FIGI. Effect of exposure to hypobaric hypoxia 16-17 h daily for

11 d

on platelet count (left) and haematocrit level (right). 0 , 0.4 atmospheres; 0. I atmosphere. Each point represents the mean of six rats. Vertical bars represent I standard deviation. Horizontal bars represent periods of exposure to hypobaric hypoxia. Asterisks indicate means significantly different from control. 7c

,L

Hypoboric hypoxio (0.4 otml H HHHHH H H H H

1

1

1

1

1

1

1

0

I

2

3

4

5

6

1

1

7 8 Time (dl

1

1

I

I

I

l

9 1 0 1 1 1 2 1 3 1 4

FIG 2. Effect of exposure to discontinuous hypobaric hypoxia on blood volume.

0.0.4 atmospheres; atmosphere; A. means significantly different from control. Each point represents six rats. Horizontal bars represent periods of exposure to hypobaric hypoxia. 0, I

days 4, 6-10 and 14. The relative blood volume increased linearly from 5.6% on day 5 to 6.3% of body weight on day 10. The overall increase in relative blood volume from day o to day

10 was

26%.

Platelet Production Rate as Measured by [ S ] Sulphate Incorporation into Platelets As shown in Table I, [3s Slsulphate incorporation into platelets of hypoxic rats was twice that of untreated animals on days 2, 3 and 4. At 7 d the [35S]sulphateincorporation was still significantly increased but had returned to control levels by day 9.

Throm bopoietic Response to Hypoxia

23 7

TABLE I. [35S]sulphate incorporation into platelets at intervals during exposure to discontinuous hypobaric hypoxia (0.4 atni)

% 35S-lncorporation

Ditratiorr qf

ESP. I

hypoxia

No. nf

into platelets

(4

rats

(xIo-~)

Untreated

12.6f 4.3

< 0.01

3

6 5 6

< 0.02

4

7

11.4 f 4.4 11.6f 2.5

2

2

Untreated 7

6 7

I)

H

11

6

P idire

5.6f 1.6'

< 0.001

4.6 f 0.9 7.1& 2.7 4.YfI.5 5.4 & 0.8

< 0.05

* I SD.

I

I

I

I

I

24

48

72

96

Time ( h )

FIG3 . Survival of 'Cr-platelcts froiii untreated donors in hypoxic or untreated recipients. 0 , Platelcts transfused into recipients on thc eighth day o f a n I I d discoiitiiiuous exposurc (16-17 h daily) to 0.4 atniosphcrcs (eight rats) ; 0.uiitrcatcd recipients (nine rats).

Sirrvival of ' Cr-Lobcllrd Plnti,lcts " Cr-labellcd platelets obtaincd from normal rats and transfused into hypoxic rats on thc cighth day of hypoxic exposure or into normal rats is shown in Fig 3. Platelet survival was the same whether tlic recipients were hypoxic or untreated. Fig 4 shows the survival of Cr-labelled platelets obtained froiii rats exposed to discontinuous hypoxia for 8 d and transfused into normal animals or into hypoxic rats on the eighth day of hypoxic exposure. Fig 4 also shows the survival of "Cr platelets from normal rats collected and labelled in parallcl with the platelets from hypoxic donors. Survival of 5 1 Cr platelets from 8 d hypoxic

Carl W. lackson and Carol C.Edwards

23 8

-0

c

80-

5

c

." 9

60 -

D

-

c

Y Y

L

40-

.-cP

0

3

! V

20-

I

I

I

I

1

24

48 Time (h)

72

96

L

I

I

I

I

24

48

72

96

Time (h)

FIG 5

FIG 4

FIG 4. Survival of S'Cr-platelets froin rats exposed to 0.4 atmospheres 16-17 h daily for 8 d. A, Hypoxic platelets transfused into recipients on the eighth day of an 11 d discontinuous exposure to 0.4 atmospheres; 0, hypoxic platelets in untreated recipients; 0 , untreated platelets in untreated recipients. Each point represents six rats. FIG 5 . Survival of slCr-platelets froin rats, exposed to 0.4 atmospheres 16-17 h daily for 11 d. A, Hypoxic platelets transfused into recipients on the eighth day of an 11 d discontinuous exposure to 0.4 atniospheres; 0 , hypoxic platelets in untreated recipients; 0 , untreated platelets in untreated recipients. Each point represents six rats.

TABLE 11. Bone marrow megakaryocyte concentration during and after

11 d

of exposure to discontinuous hypobaric hypoxia (0.4 atm)

(4

No. of rats

Untreated

I9

a 3

5 6

5.1 f 0 . I

c 0.001

5.2 f

4 7

7

4.1 f o . 4 3.0f0.3 2.8k0.6

c 0.05 < 0.001 < 0.001

Time

9 I1

I4 17

I0

8 9

Megakaryocytes/HPF

P value

6.2fo.5* 0.9

3.3fO.4

6

5.8+

6

8.4 f I .o

* I SD.

1.0