Immunohistochemical detection of apoptosis, proliferation and ...

Cell Biology International 27 (2003) 863–869

Cell Biology International www.elsevier.com/locate/cellbi

Immunohistochemical detection of apoptosis, proliferation and inducible nitric oxide synthase in rat urothelium damaged by cyclophosphamide treatment Kristijan Jezernik 1*, Rok Romih 1, Hans Georg Mannherz 2, Dasˇa Koprivec 1 2

1 Institute of Cell Biology, Medical Faculty, Lipiceva 2, 1000 Ljubljana, Slovenia Department of Anatomy and Embryology, Ruhr-University, Universita¨tsstraße 150, D-44780 Bochum, Germany

Received 24 February 2003; revised 9 June 2003; accepted 14 July 2003

Abstract The present study was conducted to investigate cell death, proliferation and inducible nitric oxide synthase (iNOS) immunoreactivity in rat urothelium within 24 h after a single intraperitoneal dose of cyclophosphamide (CP). Necrotic cells were identified predominantly in the superficial cell layer from 1 h until 6 h after CP injection, most of them desquamating from the urothelium into the lumen of the urinary bladder. Active caspase-3 immunohistochemistry revealed apoptotic cells from 12 h until 24 h after CP injection. The apoptotic index reached a peak at 18 h and then rapidly dropped. Simultaneously with the decrease of apoptosis, the proliferation index increased from 18 h until 24 h after CP treatment. Immunoreaction to iNOS was first detected at 6 h in basal and intermediate cells. Later, iNOS immunoreactivity became stronger and was present in all cell layers. Our results suggest that the destruction of rat urothelium during 24 h after CP administration is due not only to necrosis, but also to apoptosis. The first 6 h are characterised by necrotic changes and no iNOS immunoreactivity. Thereafter, apoptosis and iNOS immunoreactivity are observed within the damaged urothelium. At 24 h after CP injection, iNOS immunoreactivity is still present, but proliferation prevails over cell death, enabling the urothelium to start regeneration. 2003 Elsevier Ltd. All rights reserved. Keywords: Urothelium; Cyclophosphamide; Apoptosis; Proliferation; iNOS

1. Introduction Cyclophosphamide (CP) is inactive, per se, but it is metabolised to cytotoxic substances in the liver and possibly in the kidney (Farsund, 1976; Koss, 1967). In rats approximately 70 per cent of the metabolites of the drug is excreted in the urine within 4 h after administration of a single intraperitoneal dose (Koss, 1967). An immediate effect of CP metabolites is seen as widespread necrosis of bladder urothelium with only a few surviving cells remaining after 24 h. Nevertheless, these cells retain their ability to proliferate and re-epithelialise denuded areas (Fukushima et al., 1981; Koss, 1967; Locher and Cooper, 1970; Philips et al., 1961). Rapid epithelial regeneration follows, which leads to transient * Corresponding author. Fax: +386-1-5437681 E-mail address: [email protected] (K. Jezernik). 1065-6995/03/$ - see front matter 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S1065-6995(03)00175-6

hyperplasia by days 2 and 3 after CP injection (Fukushima et al., 1981; Koss, 1967). In CP induced destruction of rat urothelium necrotic cell death is well documented, while apoptosis has not yet been proven (Arends and Wyllie, 1991; Walker et al., 1988; Wyllie et al., 1980). However, it is known that restoration of the normal three-layered urothelium from a hyperplastic one is accompanied by apoptosis of supernumerous urothelial cells (Romih et al., 2001). In this condition, most of the urothelial cells undergo apoptosis within the superficial layer and are exfoliated into the lumen of the urinary bladder (Romih et al., 2001) and subsequently removed from the body with urine and lost for detection. Thus, it is important to identify apoptotic cells before classical features of apoptosis are present, which can be achieved by immunohistochemical detection of active caspase-3 (Hoshi et al., 1998; Stadelmann and Lassmann, 2000). Specific

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K. Jezernik et al. / Cell Biology International 27 (2003) 863–869

antisera allow a reliable detection of apoptotic cells without overlap with necrotic cell population (Armstrong et al., 1997; Srinivasan et al., 1998). Caspase-3 is an effector caspase and when activated by initiator caspases, such as caspase-8, it cleaves various cellular substrates leading to the apoptotic cell death (Bratton et al., 2000, Stadelmann and Lassmann, 2000). The immunohistochemical detection of active caspase-3 in bladder urothelium has not yet been described. A single dose of CP causes an increase in urinary nitric oxide (NO) metabolites within the first 6 h (Alfieri and Cubeddu, 2000). This is associated with the expression of calcium-independent inducible NO-synthase (iNOS) activity in the urinary bladder. Previous studies indicated a fundamental role of NO in the pathogenesis of CP-induced damage (Alfieri and Cubeddu, 2000; Alfieri et al., 2001). It is unknown whether urothelial cells are capable of iNOS expression and NO synthesis. Different studies demonstrate that the presence of a functionally active iNOS is a crucial prerequisite for normal wound healing and inhibition of iNOS causes a substantial reduction in the number of proliferating keratinocytes and fibroblasts (Shi et al., 2001; Stallmeyer et al., 1999). NO acts also as a powerful inducer of apoptosis or of necrosis in some cells, but represents an equally powerful protection from cell death in others (Kro¨ncke et al., 2001; Suschek et al., 1999; Yue et al., 2001). This indicates that iNOS may play an important role in cell death and proliferation of urothelial cells exposed to CP metabolites. The object of this study was: first, to investigate whether urothelial cells undergo apoptosis; second, to observe the dynamics of necrosis, apoptosis and proliferation; and third, to evaluate the correlation between these three processes and iNOS expression during CP-induced destruction of the rat urothelium. 2. Materials and methods

and iNOS immunohistochemistry and four animals were used for PCNA detection. Only dilated bladders were further processed, which is considered the prerequisite for estimation of hyperplasia. Two syringes were introduced into each bladder. One was used to remove and to measure the volume of the urine and the other syringe was used simultaneously to introduce the same volume of appropriate solution, ensuring that bladders retained their expanded form. 2.2. Electron microscopy Urine was replaced by the fixative (4.5% paraformaldehyde and 2% glutaraldehyde) and after 15 min bladders were removed from animals, dissected in the same fixative and fixed for additional 3 h. Samples were postfixed in OsO4, dehydrated in ethanol and embedded in Epon. 2.3. Active caspase-3 immunohistochemistry Urinary bladders were filled with 4% paraformaldehyde in PBS buffer, removed after 15 min and embedded in paraffin. The paraffin blocks were used for active caspase-3 or inducible NOS immunohistochemistry. For active caspase-3 detection, paraffin sections were deparaffinised, washed in TBS buffer and non-specific labelling was blocked by 10% normal goat serum in TBS. Sections were incubated with a polyclonal antibody against active caspase-3 (R&D Systems) diluted with 1% BSA in TBS. Alkaline phosphatase-conjugated goat anti-rabbit antibodies were applied, followed by Dako fuchsin substrate-chromogen system (DAKO Corporation Carpinteria, USA). Sections were counterstained with haematoxylin and examined with a Nikon, Eclipse TE300 microscope. As negative controls, incubations with anti-active caspase-3 antibodies were omitted.

2.1. Animals and treatment

2.4. PCNA (proliferating cell nuclear antigen) immunohistochemistry

Animal experiments were approved by the Veterinary Administration of the Slovenian Ministry of Agriculture, Forestry and Food according to the Law for Animal Health Protection and the Introductions for Granting Permits for Animal Experimentation for Scientific Purposes. Adult male, 5 weeks old F344 rats received a single intraperitoneal injection of CP (Sigma) at doses of 100 mg per kg of body weight, since this dose ensures reversible regenerative hyperplasia of the urothelium (Fukushima et al., 1981). Ten rats were sacrificed at each time point, i.e. at 1, 6, 12, 18 and 24 h after CP injection. Ten control rats received injections of 0.3 ml distilled water without CP. From each group bladders of two animals were processed for electron microscopy, four animals were used for active caspase-3

Urinary bladders were first filled with 0.9% NaCl, which was immediately replaced by OCT embedding medium (Tissue-Tek; Miles). Bladders were removed from animals and frozen in liquid nitrogen. On cryostat sections, endogenous peroxidase activity and nonspecific labelling was blocked by incubation with 3% H2O2 in methanol and 2.5% BSA in PBS, respectively. Tissue sections were incubated with a monoclonal antibody against PCNA (PC-10, DAKO) diluted with 0.1% BSA in PBS. Biotinylated rabbit anti-mouse antibodies were applied, followed by ABC/HRP complex (DAKO A/S, Denmark). Peroxidase activity was demonstrated by 3,3#-diaminobenzidine and sections were counterstained with haematoxylin. As negative controls, incubations with anti-PCNA antibodies were omitted.


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2.5. Inducible NOS immunohistochemistry For detection of iNOS, paraffin sections were microwave heated for antigen retrieval. Endogenous peroxidase activity was blocked by incubating the sections in 3% H2O2 for 15 min. After washing in PBS, sections were pre-incubated in normal swine serum (DAKO; 1/5) and then incubated with the primary polyclonal antibody against iNOS (BD Transduction Laboratories; 1/500) overnight at 4 (C. After washing in PBS, biotinylated goat anti-rabbit immunoglobulins (DAKO; 1/200) were applied for 30 min and then the incubation with ABC/HRP complex followed. After the standard 3,3#-diaminobenzidine development procedure, sections were counterstained with haematoxylin. As negative controls, incubations with primary antibodies were omitted. 2.6. Quantitative evaluation Using active caspase-3 and PCNA staining, we determined the apoptotic index and proliferation index, respectively. Four treated rats were used for each staining at each time point (1, 6, 12, 18 and 24 h after CP injection) plus 4 control rats. From each bladder sections throughout whole circumference were made at three different positions (base, middle and dome of the bladder). All urothelial cells were counted which always made at least 1000 cells per urinary bladder. Apoptotic and proliferation indices were defined as a percentage ratio of positively stained cells to the total number of counted cells. The values were expressed as meanstandard deviation. Differences between the means of treated rats and of the controls were tested by two-sided Student’s t test.

3. Results In the untreated control rats the urothelium consisted of three cell layers. One hour after the injection of CP it was already possible to see necrosis and detachment of individual superficial cells. Six hours after the injection extensive urothelial damage was observed. In many areas, superficial cells were detached and the intermediate cell layer was exposed to urine. At 12 h, the urothelium was one to two cell layers thick and there were numerous areas completely depleted of cells and thus the basal lamina was exposed. Eighteen hours after the injection, the urothelial damage was similar to that at 12 h. Additionally, numerous cells with apoptotic features were seen at this time point. At 24 h after the injection some areas of the urothelium were again composed of three cell layers. Necrotic cells were characterised by ruptured plasma membranes and swelling of the cytoplasm and nucleus.

Fig. 1. TEM micrograph. Necrotic cell with swollen cytoplasm and nucleus in the superficial layer of the urothelium at 6 h after injection of CP. Scale bar, 5 µm.

They were located predominantly in the superficial cell layer, most of them desquamating from the urothelium into the lumen of the urinary bladder, from 1 h until 24 h after CP application, but most intensely between 1 and 6 h (Fig. 1). Apoptotic cells, characterised by classical morphological features of apoptosis, were appearing in the basal, intermediate and superficial layer of the urothelium from 12 h until 24 h after CP administration (Fig. 2a). Apoptotic cells were also seen detaching from the luminal surface of the urothelium and in the lumen of the urinary bladder (Fig. 2b). In controls, at 1 and 6 h after injection of CP active caspase-3-positive cells were not detected. At 12 h, a substantial elevation of apoptotic index was detected (Fig. 3), with positive cells appearing in the basal, intermediate and superficial cell layer (Fig. 4a). Apoptotic cells were seen protruding from the superficial cell layer into the lumen of the urinary bladder (Fig. 4a). The apoptotic index reached a peak of 5.1% at 18 h (Fig. 3), with positive cells appearing in all layers and in the lumen of the urinary bladder (Fig. 4b). At 24 h, the apoptotic index was still slightly higher (0.13%) than in controls (0.0%). In controls, PCNA-positive cells were rare (the proliferation index in Fig. 3) and exclusively limited to the basal cell layer. At 1 h after injection of CP, the proliferation index was slightly raised and it slowly increased from 6 until 18 h (Fig. 3). The PCNA reaction was seen most abundantly in nuclei of the basal cell layer, but many were also stained in intermediate and

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Fig. 2. TEM micrograph. (a) Apoptotic cell with condensed chromatin and cytoplasm in the basal layer of the urothelium at 24 h after injection of CP. Basal lamina is seen (arrow). Scale bar, 5 µm. (b) Apoptotic bodies and a mass of cell debris in the lumen of the urinary bladder 18 h after injection of CP. Scale bar, 5 µm.

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ficial iNOS-positive cells were also seen. Similar results were observed at 12 h, with most of the urothelium being stained in all cell layers. At 18 and 24 h, the reaction was very strong in the whole urothelium and also in some detached urothelial cells in the lumen of the urinary bladder (Fig. 6b).

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Fig. 3. Apoptotic and proliferation indices of the urinary bladder urothelium after single intraperitoneal CP injection. Standard deviations are indicated on bars. P values show statistical difference between CP-treated and control groups of rats. Note that apoptotic index was zero in controls and after 1 or 6 h after treatment.

even superficial layers of the urothelium. Thereafter, the proliferation index rapidly increased and reached the value of 5.1% at 24 h (Fig. 3), with PCNA-positive cells prevailing in the basal cell layer (Fig. 5), but also appearing in intermediate and superficial cell layers. The immunohistochemical detection of iNOS showed no reaction in controls and at 1 h after injection of CP. At 6 h, some areas of urothelium were not labelled, while most of the urothelium was stained within the basal and intermediate cell layer (Fig. 6a). Some super-

CP-induced lesions of the urinary bladder of the rat are characterised by nearly complete detachment of the urothelium, which is thought to be due to necrosis and desquamation of urothelial cells (Farsund, 1976; Fukushima et al., 1981; Koss, 1967). The first response to the CP metabolites undoubtedly is necrosis, with necrotic cells appearing predominantly in the superficial cell layer from 1 until 6 h after CP injection. These observations indicate that the toxic stimulus leading to necrosis comes directly from urine (Koss, 1967; Philips et al., 1961). It is generally assumed that high concentrations of toxins lead to necrosis whilst mild degrees of toxic assaults lead to apoptosis (Duvall and Wyllie, 1986). We have found that during the initial exposure of urothelial cells to CP metabolites not only necrosis, but also apoptosis leads to desquamation and loss of cells from the urothelium. Apoptotic cells appeared in large number within the urothelium already at 12 h after CP treatment. Thereafter the apoptotic index further increased to a peak of 5.1% after 18 h and suddenly dropped to the value of 0.1% after 24 h, clearly confirming that during destruction of rat urothelium apoptosis follows necrosis and


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Fig. 4. Active caspase-3 immunohistochemistry. (a) Active caspase-3-positive cells (red) in basal and superficial cell layer at 12 h after injection of CP. Positive cells are protruding into the lumen of the urinary bladder. Scale bar, 10 µm. (b) Active caspase-3-positive cells (red) in all cell layers of the urothelium, which is still in contact with lamina propria (LP), and in the lumen of the urinary bladder (L), where a mass of cells and cell debris is seen, at 18 h after CP injection. Scale bar, 10 µm.

Fig. 5. PCNA immunohistochemistry. PCNA-positive cells (brown nuclei) in the basal layer of urothelium 24 h after CP injection. L marks the lumen of the urinary bladder. Scale bar, 10 µm.

also plays an important role in cell elimination. We have demonstrated this by active caspase-3 immunohistochemistry and confirmed this by electron microscopy. Results of our study show that immunohistochemistry of active caspase-3 is a reliable tool in identifying early apoptotic cells in the urothelium. Both methods show that apoptotic cells appear in all cell layers, indicating that cells of all urothelial layers are susceptible to apoptotic stimuli. Apoptosis often occurs around the areas of tissue necrosis and its role could be to remove the cells that have been damaged but not underwent necrosis. Different previous studies demonstrated that the 3Hthymidine (3H-TdR) labelling index increases during first 24 h after injection of CP (Farsund, 1976; Kunze et al., 1984). It is unknown when proliferation activity

Fig. 6. Inducible NOS immunohistochemistry. (a) Positive reaction (brown) in basal and intermediate layers of the urothelium 6 h after CP injection. Scale bar, 10 µm. (b) Positive reaction in all cell layers and in detached urothelial cells 18 h after CP injection. Scale bar, 10 µm.

starts to increase and which cells possess the ability to divide. Our results of PCNA immunohistochemistry showed that the proliferation index increased slowly from 6 until 18 h after CP injection, and very rapidly

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from 18 until 24 h, with a peak value of 5.1% at 24 h. PCNA-positive cells were most abundant in the basal layer, but also appeared in the intermediate and even superficial layers in contrast to control urothelium in which only basal cells divide. It therefore seems that after CP application urothelial cells from all layers proliferate, while the basal cells respond most rapidly to proliferative stimuli. Our previous study indicated that undifferentiated urothelial cells from hyperplastic urothelium express epidermal growth factor receptor (EGFR) over their entire plasma membrane and are therefore responsive to EGF from urine (Romih et al., 2001). A similar mechanism may be involved in the induction of proliferation of undifferentiated urothelial cells exposed to urine within 24 h after CP administration. Our results showed an inverse correlation between opposing processes of proliferation and apoptosis during the time-period from 18 to 24 h after injection of CP. Apoptosis during that time was reduced while proliferation increased, which might enable the urothelial regeneration and development of a hyperplastic urothelium. In normal rats, no immunoreactivity to iNOS was noted in bladder urothelium. However, strong immunoreactivity was described in rat urothelium after lipopolysaccharide (LPS)-induced endotoxic shock (Persson et al., 1999). Current evidence shows that iNOS activity is increased in bladders of CP-treated rats, where it plays an important role in increasing the local production of NO (Alfieri and Cubeddu, 2000; Alfieri et al., 2001). In the present study we established that after CP treatment iNOS immunoreactivity is present in all urothelial cells. At 6 h after CP injection the iNOS labelling was found predominantly in cells located in the basal and intermediate layers of the urothelium. Thereafter immunoreactivity to iNOS extended over the whole urothelium. These results show that iNOS is not labelled during necrosis of urothelial cells after CP injection. With increased apoptosis, iNOS immunoreactivity is also enhanced and it stays the same when proliferation prevails over cell death. We do not know whether iNOS expression contribute to apoptosis or does it on the contrary contribute to protection and urothelial regeneration. Further studies are needed to give insight into this question. In summary, the present study revealed that CP induced destruction of rat urothelium is accompanied not only by necrosis but also by apoptosis. Immunohistochemical detection of active caspase-3 is a valuable tool for identification of apoptotic cells in rat urothelium. Proliferation occurs already during destruction of the urothelium after a single CP injection. Correlation between iNOS immunohistochemistry and an increase of apoptosis and proliferation of urothelial cells was observed, but the functional role of iNOS in regulating

apoptosis and proliferation of urothelial cells remains to be established.

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