Eur J Gen Med 2004; 1(4): 15-21

ORIGINAL ARTICLE

A STEREOLOGICAL STUDY ON AZUROPHILIC AND SPECIFIC GRANULES IN OF PATIENTS WITH SCHIZOPHRENIA BEFORE AND DURING ANTIPSYCHOTIC TREATMENT

Ibrahim Mohammed Inuwa1, Nona Viernes 2, Ziad Zaidan2

Sultan Qaboos University, Departments of Human and Clinical Anatomy1 and Behavioural Medicine2

Antipsychotic drugs, frequently used for treating schizophrenia, have many unpleasant and sometimes life threatening side effects. Some of these side effects, such as agranulocytosis cannot be explained satisfactorily on the basis of the drugs’ pharmacological actions. The first indication of this event is neutropenia which then progresses to full blown agranulocytosis. This study was carried out to quantitatively assess maturity in patients on antipsychotic drugs using the appearance of their granules on electronmicroscopy. Blood samples were collected from schizophrenic patients (n = 20) before and six weeks after starting treatment with two types of Antipsychotic drugs (Risperidone and Chlorpromazine) and from healthy subjects (n = 21) having no history of antipsychotic drugs medication and processed for transmission electron microscopy. Volume density of azurophilic (electron-dense) granules in patients after treatment was found to be greater than both before treatment and in control group. The volume weighted mean volume of granules was significantly greater in cells of patients after treatment. There were significantly more neutrophils with fewer than three lobes of nuclei in patients after treatment than in controls or before treatment. These findings suggest neutrophil cellular immaturity in the patients during 6 weeks of treatment even though there was no haematological evidence of neutropenia. It is suggested that immaturity of neutrophils manifests before any evidence of neutropenia. Key words: Schizophrenia, antipsychotics, neutrophils

INTRODUCTION as sedation, dystonia and muscle stiffness, Schizophrenia is a chronic debilitating tremors, elevated prolactin levels, and psychiatric disorder. Its etiology is not weight gain (6). Many patients with well understood and probably consists negative symptoms of schizophrenia do of several separate illnesses. Symptoms not respond to conventional antipsychotic include disturbances in thoughts, affects, drugs which may necessitate the use of and perceptions and difficulties in atypical antipsychotic ones (7). Although relationships with others. The hallmark the use of atypical antipsychotic drugs is symptom of schizophrenia is the experience associated with marked improvement in the of auditory hallucinations. The mainstay of symptoms of schizophrenia, their use is treatment on this condition is by the use of linked to the increased risk of development of antipsychotic drugs (1-3). These drugs fall agranulocytosis, which can occur in about 1% into two categories; conventional ones such of patients (8,9). This side effect is usually as chlorpromazine and unconventional ones unpredictable and consequently, frequent such as clozapine. The mechanism of action monitoring of WBCs is required in such of these drugs is believed to be through patients (10). their anti-dopaminergic activity specifically During human neutrophil differentiation, dopamine D2 receptor antagonism (4,5). large portions of the genome condense and Conventional antipsychotic drugs associate with the nuclear envelope to form are associated with adverse effects, such filament-like structures. As a result, the nucleus of the mature neutrophil typically Correspondence: Ibrahim Mohammed Inuwa consists of a linear array of three or four Department of Human and Clinical Anatomy, Sultan Qaboos University, PO Box 35. Al Khod 123. Muscat, lobes joined by thin, DNA-containing Sultanate of Oman filaments. Nuclear segmentation is therefore Telephone: +968 515176, Facsimile: +968 513419 an indicator of maturity of neutrophils (11). E-mail: [email protected] 16 Inuwa et al.

1a 1b

1c 1d Figure 1. Neutrophils having varying nuclear lobation. (a) 2 lobes, (b) 3 lobes, (c) 4 lobes, (d) 5 lobes. Bilobed cells are immature while 3-5 lobed cells are mature neutrophils.

At electronmicroscopy, the appearance of This study used light and neutrophil granules are used as indicators electronmicroscopic stereological methods to of maturity as well. There are two types of evaluate maturity of neutrophils in patients granules in the neutrophil based on their on treatment with antipsychotic drugs before electron density; light staining granules are any laboratory evidence of the onset of predominantly found in mature cells whereas neutropenia in such patients. dark staining (azurophilic) granules are predominantly found in immature cells (12). MATERIAL AND METHODS Stereological morphometry has been used Experimental design by many workers on normal and pathological Patients recruited in the study were newly tissue to obtain quantitative structural admitted with the diagnosis of schizophrenia. information that may have physiological or The diagnosis was made using the DSM- biochemical correlates (13-15). The majority IV criteria (Am Psych Assoc. 1994). After of stereological parameters are ratios such as appropriate consent from the next of kin was volume density, or absolute quantities such obtained the patients were clinically examined as total volume, surface area, length etc (16). for obvious evidence of infection. Stereological morphometry is a powerful Blood samples taken for total blood counts tool because it provides a three-dimensional (CBC). Those patients found to have evidence information which makes interpretation of of active infection were excluded from the functional morphology more effective study. Within a period of six months, 20 17 Azurophilic and specific granules in Schizophrenia

rpm using a bench top refrigerated micro centrifuge (Sanyo Hawk). The cell pellet was then postfixed in 1% osmium tetroxide dehydrated in increasing acetone series, and embedded in Araldite. Semithin sections obtained with a Leica ultracut microtome were stained with toluidine blue so as to identify appropriate cell types. Silver sections were then obtained, mounted on copper grids, double-stained with uranyl acetate and lead citrate (18) and observed in a Zeiss EM900 transmission electronmicroscope.

Stereology All stereological estimates were obtained using Histometrix software (Kinetic imaging Ltd) attached to an IBM personal computer. Measurements were made on digitized Figure 2. Transmission electronmicrograph images of neutrophils obtained from the of neutrophil showing granules staining. electronmicroscope. volume density (}) azurophilic or immature granules,(→); was estimated using the point counting specific granules. method by randomly throwing test points on the micrograph (19). Volume weighted mean volume of granules was estimated using the patients satisfied the inclusion criteria and point-sampled intercept method (20). Data were used for the study. The control group was reported as group mean +/- standard were a similar number of age-matched error of the mean. Difference between patient subjects who were not taking any sort of groups was analysed using paired student t medication at the time of the study. At day 0, test while difference between patient groups (before the start of treatment) 2 millilitres of and controls was analysed using ANOVA with venous blood was obtained from each patient. Neuman multiple comparison post test. Mean Patients were then treated with clozapine difference was considered to be statistically (300 mg/day) or risperidone (6 mg/day) for significant if P was found to be less than a period of 4 to 6 weeks. At the end of 6 0.05. weeks 2 millilitres of venous blood was again obtained. Complete blood count (CBC) in RESULTS patients was monitored routinely every week Mean numbers of nuclear lobes in throughout the six weeks of treatment. neutrophils are shown in table 1. Amongst the control group, nuclei of 90.4 % of cells Light microscopy had between 3-5 lobes whilst 9.3 % have less Two millilitres of venous blood sample than 3 lobes. In the untreated patients, 91.9 obtained was placed in heparinised containers % of cell have nuclei that have between 3-5 from each patient and control subject. A drop lobes and 8.1 % have less than 3 lobes (Fig of the heparinised blood was placed on a 1). After six weeks of treatment, 29.6 % of glass slide and a thin blood film was made. the neutrophil population of the patients were The blood film was fixed with 70% acetone found to have 3-5 lobed nuclei and 70.3 % and stained with Giemsa stain. One hundred had less than 3 lobed nuclei (p<0.05). The systematic random sample of neutrophils per total and differential white blood cell counts patient were evaluate for nuclear lobation in all the three groups were within normal according to Arneth Cooks criteria (17). range for the general population and were not Transmission electronmicroscopy significantly different from one another (see One millilitre of the blood sample was Table 1). placed in a 2ml tube centrifuged down, Volume density of electron dense granule the white blood cell layer (buffy coat) (Fig 2) populations in controls was 0.0408 was removed, and suspended in buffered ± 0.001 (s.e.m), whilst that of light staining Karnovski fixative (Ph 7.26) for 30 minutes. granules was 0.0714 ± 0.001 (s.e.m). Volume The suspended cells were converted into density of electron dense granule populations a pellet (1mm3) by centrifugation at 1500 was significantly higher in treated group than 18 Inuwa et al.

A B 0.100 p<0.001

0.075 0.100 p>0.05

p>0.05 0.075 0.050 p<0.001 0.050

0.025 0.025

0.000 0.000 Control Untreated Treated Control Untreated Treated Figure 3. A-Scater plot of volume density of electron dense granules in neutrophils. B-Scater plot of volume density of light staining granules in neutrophils. in untreated group and control (p<0.001). volume regardless of type was 2.223 ± There was no significant difference (p>0.05) 0.09 (s.e.m) µm3 in control group, 2.167 in volume density of both groups of granule ± 0.05 (s.e.m) µm3 in untreated group and populations between controls and untreated 2.706 ± 0.11 (s.e.m) µm3 in treated group. groups (Fig 2). Mean ratio between electron Granule volume weighted mean volume was dense and light staining granules in each cell significantly greater (p < 0.001) in treated was 0.526 ± 0.19 (s.e.m) in the control group. patients as compared to untreated (Fig 4). This ratio was significantly increased (p < 0.001) in treated group when compared with DISCUSSION controls and untreated groups. No significant Our data has demonstrated that total white difference (p > 0.05) was found between blood cell count and neutrophil count in controls and untreated group (Fig 3). untreated patients and those treated for six Average granule volume weighted mean weeks were not significantly different from

4

p<0.001

3

2

1 p>0.05

0 Control Untreated Treated

Figure 4. A- Box plot showing granule ratio (dense:light) in neutrophil populations in the three study groups. 19 Azurophilic and specific granules in Schizophrenia

5

4

3

2

1

0

Figure 4. B-Box plot showing volume weighted mean volume of granules in neutrophil populations in the three study groups.

controls. However, a significant percentage explain the observed neutrophil morphology, of those neutrophils in treated patients these findings could be a consequence of drug have bilobed nuclei. This is an indication treatment. An equally important question to of immaturity and suggests that the bone ask is if these effects on neutrophils are due marrow is releasing cells before the nuclei to drug treatment, then, what is the likely are completely segmented. Such mechanism involved? response is usually secondary to an increase The mechanism of action of antipsychotic in demand due either peripheral neutrophil drugs used in treatment of schizophrenia is destruction or malignant transformation believed to be through their antidopaminergic (21,22). effect. It could it be possible that their anti Neutrophils have two distinct granule dopaminergic activity is the basis of the populations; azurophilic staining granules observed neutropenic effect. Sookhai et which are large and appear as electron dense al (24) have demonstrated the presence of granules on elecronmicroscopy, and specific dopamine receptors in neutrophil populations granules which are small and appear as light of normal human volunteers. These receptors staining granules on elecronmicroscopy. are the D2 family (D2,3,4) which are known to Azurophilic (primary) granules are more be the sites of activity of antipsychotic drugs. abundant in immature cells than specific However, in the same report, the investigators (secondary) granules (23). Our data on granule found that stimulation of the receptors with volume density, granule ratio, and absolute dopamine agonists induced apoptosis in volume weighted mean volume correlated neutrophils. Therefore the immaturity of positively with the light microscopy finding neutrophils observed in our study could not of immaturity. satisfactorily be explained on the basis of In the absence of any source of infection, drug-receptor interaction – if anything, the or other pathologic condition that could antipsychotic drugs being antidopaminergic

Table 1. Mean number of lobes in the nuclei of neutrophil leucocytes, total WBC count and total neutrophil count in the three study groups n Mean lobe WBC(x109)/l) Neutrophil(x109)/l)

Untreated 20 2.69 ± 0.1 5.64 ± 0.71 3.51 ± 0.39 Treated 20 *2.21 ± 0.23 6.37 ± 1.89 3.64 ± 1.61 Controls 21 2.75 ± 0.08 5.96 ± 0.67 3.33 ± 0.46 20 Inuwa et al. should not cause neutropenia! Loeffler, et al (25) have suggested that Acknowledgement the observed neutropenia and subsequent We are grateful to the Sultan Qaboos University for funding this project, and to the nurses of psychiatric agranulocytosis is due to enhanced apoptosis ward of Sultan Qaboos University hospital for assistance of blood neutrophils resulting from enhanced with blood sample collection. expression of the pro-apoptotic proteins Bax and Bik and from a decreased expression REFERENCES of the anti-apoptotic BCl-X(L) mRNA. The 1- American Psychiatric Association: antipsychotic drug commonly associated with Diagnostic and statistical manual of the highest risk of agranulocytosis – clozapine mental disorders, 4th ed, Washington, DC, is known to undergo bioactivation to a 1994 chemically reactive nitrenium ion. This ion 2- Mortimer AM. Antipsychotic treatment in has been suggested by Williams et al. (26,27) schizophrenia: atypical options and NICE to be the agent responsible for neutrophil guidance. Eur J Psychiatry 2003;18:209-19 apoptosis through a mechanism of cell surface 3- Kane JM. The current status of neuroleptic haptenation and glutathione depletion. therapy. J Clin Psychiatry 1989;50:322-8 Another possible explanation for 4- Pickar D. Perspectives on a time- immaturity of neutrophils could be due dependent model of neuroleptic action. to direct intracellular accumulation and Schizophr Bull 1988;14(2):255-68 subsequent cytotoxicity. It could be that 5- Josselyn SA, Miller R, Beninger RJ. the drugs preferentially accumulate inside Behavioral effects of clozapine and neutrophils causing cell death which leads to dopamine receptor subtypes. Neurosci a reactive myeloblastic response of releasing Biobehav Rev 1997;21(5):531-58 more immature cells into the circulation. A 6- Guze SB. The psychiatric drug handbook, structure-activity relationship model using 2nd ed, St. Louis, Mosby 1995 physicochemical parameters such as lipid 7- Jibson MD, Tandon R. New atypical solubility, ionic charge and molecular weight antipsychotic medications, J Psychiatr of drugs developed and used to determine Res 1998;32:215-28 where substances will accumulate inside cells 8- Beers MH, and Berkow R. The Merck (28) has shown that, antipsychotic drugs have manual of diagnosis and therapy. 17th ed. the properties that will make them accumulate 2004;15:193 inside mitochondria of cultured fibroblasts. 9- Iverson S, Zahid N, Uetrecht JP. It could well be possible that these drugs Predicting drug-induced agranulocytosis: accumulate intracellular preferentially in characterizing neutrophil-generated neutrophils causing mitochondrial toxicity metabolites of a model compound, DMP and subsequent cell death. Indeed it has been 406, and assessing the relevance of an in observed that mitochondria of patients treated vitro apoptosis assay for identifying drugs with antipsychotic drugs were swollen and that may cause agranulocytosis. Chemico- had disrupted cristae – features indicating Biological Interactions 2002;142(1-2): direct injury (29). 175-99 In conclusion, although it has long been 10- Bernstein JG. Handbook of drug therapy acknowledged that the use of antipsychotic in psychiatry, 3rd ed, St. Louis: Mosby drugs is associated with a risk of unexplained year book, 1995:400 agranulocytosis, it is suggested here that a 11- Sanchez JA and Wangh LJ. New likely mechanism of this effect is through insights into the mechanisms of nuclear direct selective neutrophil toxicity possibly segmentation in human neutrophils. J involving mitochondrial destruction and Cellular Biochemistry 1999;73(1):1-10 subsequent cell death. The earlier indication 12- Brederoo P, Van der Meulen J, Mommaas of this effect could be the presence of Kienhuis AM. Development of the granule immature neutrophils in peripheral blood population in neutrophil without associated evidence of neutropenia. from human bone marrow. Cell and Tis It is therefore suggested that patients treated Res 1983;234(3):469-96 with antipsychotic drugs particularly the 13- Mayhew TM. Striated brush border of atypical ones should have their neutrophil intestinal absorptive epithelial cells: granulocytes assessed for maturity even stereological studies on microvillous if there is no haematological evidence of morphology in different adaptive states. J neutropenia. Electron Microscopy Technique 1990;16: 45–55 21 Azurophilic and specific granules in Schizophrenia

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