ApoSense: a novel technology for functional molecular imaging of cell death in models of acute renal tubular necrosis

Purpose Acute renal tubular necrosis (ATN), a common cause of acute renal failure, is a dynamic, rapidly evolving clinical condition associated with apoptotic and necrotic tubular cell death. Its early identification is critical, but current detection methods relying upon clinical assessment, such as kidney biopsy and functional assays, are insufficient. We have developed a family of small molecule compounds, ApoSense, that is capable, upon systemic administration, of selectively targeting and accumulating within apoptotic/necrotic cells and is suitable for attachment of different markers for clinical imaging. The purpose of this study was to test the applicability of these molecules as a diagnostic imaging agent for the detection of renal tubular cell injury following renal ischemia.
Introduction
Acute renal failure (ARF) that results from ischemic or toxic insults to the kidney is usually referred to clinically as acute tubular necrosis (ATN), where tubular damage and altered glomerular hemodynamics may coexist or even lead to each other [1–3]. In some cases of either human ARF [4, 5] or experimental models of renal injury [6], a lack of correlation between histological evidence of injury and renal function has been found, and the relative contribution of functional versus structural changes to the evolving renal dysfunction has not been unequivocally substantiated. Since ATN is a dynamic clinical condition, rapidly evolving from the incipient to the established phase, its early identification is critical. However, the various currently available methods of clinical and laboratory assessment are insufficient to differentiate between ischemic ARF, resulting in acute tubular dysfunction secondary to cell injury, and prerenal azotemia, characterized by decreased glomerular filtration but no epithelial cell injury. The inability to distinguish between these situations leads to long disputes over the pathogenesis, diagnosis, and management of evolving renal failure. Renal biopsy provides limited data because of the small sample size and the very focal pattern of tubular injury [2, 7]. In addition and most importantly, it is impractical in the human syndrome. Functional data in early phases are also of little help, because prerenal azotemia becomes an important consideration at that time, and only the evolution of the clinical situation really defines the nature of the problem [8, 9]. The utilization of specific urinary biomarkers of tubular injury, such as KIM-1, is not yet considered valid or sufficiently sensitive to establish ATN [10, 11]. Moreover, these tests remain qualitative rather than quantitative markers of injury. Since therapeutic interventions might differ at various points in the evolution of ATN, advanced techniques are required to noninvasively follow the dynamic status of tubular injury. Furthermore, a technology that enables imaging endpoints, instead of time-consuming dissection and detection of renal parenchymal cellular damage, would be highly valuable.
Multiple lines of evidence suggest that both apoptotic and necrotic cell death are important pathogenic mechanisms in acute renal failure [12, 13], where renal parenchymal cells are the primary target of a broad spectrum of inciting factors, ranging from ischemic and nephrotoxic agents to endotoxemia. Apoptotic cell death is a feature of both ischemia–reflow injury [14] and radiocontrast-induced acute renal failure [15], and is a documented event in transplant biopsy as well [16]. The role of apoptosis in sepsis syndrome has not been adequately explored, but there is rapidly developing evidence to suggest that increased apoptotic processes may play a determining role in the outcome of sepsis syndrome [17, 18]. Although apoptosis is recognized as playing a role in these conditions, there is a lack of sensitive markers to monitor programmed cell death in association with disease progression or regression.
Recently, we developed a family of small molecule compounds, ApoSense, capable of discriminating between vital and apoptotic cells. ApoSense molecules are small non-peptidic fluorescent compounds that are capable of selective targeting, binding, uptake, and accumulation within apoptotic and/or necrotic cell cytoplasm, from the early stages of the death process. Our preliminary results from different animal models associated with apoptosis showed that the recognition of damaged cells by ApoSense is universal, irrespective of the cell type or the apoptotic/necrotic trigger. Indeed, examined in multiple animal models, ApoSense was proven to be a highly sensitive and specific sensor for in vivo detection of neoplastic, hepatic, myocardial, atherosclerotic, and neuronal apoptotic and necrotic cell death (in preparation). In the present study the potential of both fluorescent and radiolabeled derivatives of one of the ApoSense compounds, didansyl cystine (DDC), was explored as a diagnostic imaging agent for detection of renal tubular injury in three different in vivo animal models of ATN: renal warm ischemia/reperfusion, radiocontrast-induced distal tubular necrosis, and cecal ligature/perforation-induced sepsis. These different models illustrate the applicability of ApoSense as a diagnostic imaging agent enabling real-time accurate detection of renal parenchymal damage of various types, and evaluation of its distribution and magnitude.
Materials and methods
Male Sprague-Dawley rats weighing 180–250 g and 8- to 10-week-old C57Bl mice (Harlan Laboratories, Jerusalem, Israel) were used for these experiments. All experimental protocols were performed according to the Guiding Principles for Research Involving Animals, and approved by the local Animal Care Committee; the experiments described in this manuscript also comply with the current laws of Israel.
ApoSense synthesis and radiolabeling
The ApoSense compound used in the present study was N,N′-didansyl-L-cystine (DDC). Both its inherent fluorescent characteristics and its radiolabeled derivative (labeled with 3H radionuclide) were used for identification and quantification of apoptotic cells.
For DDC synthesis, dansyl chloride (675 mg) and cystine (240 mg) were dissolved in water/acetone solution with potassium carbonate (550 mg), and after 1.5 h at room temperature, the aqueous mixture was extracted with ethyl acetate. The crude DDC was dissolved in 5% sodium carbonate, washed with ether, and reacidified to pH 3 to afford a yellow precipitate. The product was analyzed using 1H-NMR.
Labeling of DDC with 3H was performed by consecutive addition of 0.16 mg cystine (0.65 μmol), 0.57 mg sodium carbonate (5.3 μmol), and 37 MBq (1 mCi) of 3H-dansyl chloride (Vitrax) at a concentration of 1.4×10−4 mmol. The product was purified using a Bond-Elut C-18 cartridge eluted with 25% acetonitrile in 0.1% TFA in water, then lyophilized and dissolved in 1 ml of NaPPi buffer (0.1 M, pH=7.4). Radiochemical purity was assessed by thin-layer chromatography.
Evaluation of ApoSense binding to apoptotic cells in vitro
Human adult T-cell leukemia Jurkat cells (clone E6-1) and cervical carcinoma HeLa cells (CCL-2.2), were purchased from ATCC (Rockville, MD, USA) and grown in Dulbecco’s modified Eagle’s medium with 10% fetal calf serum at 37°C in 5% CO2. For induction of apoptosis in the Jurkat cell sample, 1×106 cells/ml were treated with IgM anti-Fas (CD95) antibody, clone CH11 (Medical and Biological laboratories, Japan) at a concentration of 0.1 μg/ml for 120–180 min. In additional studies, before incubation with anti-Fas antibody, Jurkat cells were subjected to 50 μM of caspase inhibitor Z-VAD-FMK in 0.05% DMSO (Enzyme System Products, Dublin, CA). For apoptosis induction in HeLa cells, growing cells were treated with staurosporin (STS; 250 nM; Sigma) for 18 h. At the end of the incubation period, cells were harvested by trypsinization (Trypsin-EDTA, Bet-Haemek, Israel) and collected by centrifugation.
For assessment of apoptosis, both fluorescence microscopy and flow cytometry were employed. Trypsinized HeLa cells were incubated with DDC (50 μM) and annexin V-FITC (Phosphatidyl Serine Detection Kit, IQP-116F) for 20 min and the staining pattern was evaluated under a fluorescence microscope (BX51TF; Olympus Optical.Co., Ltd., UK) equipped with relevant filters. For DDC staining, excitation at 365 and emission at 420 (band pass) nm was employed, whereas for FITC detection, excitation was at 488 nm and emission at 530 nm. Apoptotic Jurkat cells were detected by flow cytometry after double staining with annexin V-FITC and PI or with DDC and PI. Briefly, 30 μl of cell suspension (1×106 cells/ml) was diluted in 300 μl Hepes buffer (10 mM Hepes and 140 mM NaCl, pH 7.4) containing annexin V-FITC/PI or 50 μM of DDC/PI. Following 40 min of incubation at room temperature, the cells were subjected to analysis using FACS Vantage VE (BD Biosciences, San Jose, CA) and CellQuest software. A total of 10,000 events were collected for each sample.
Evaluation of ApoSense uptake into apoptotic/necrotic cells in vivo
The detection of renal parenchymal cell death by ApoSense was assessed in three animal models of ATN, generally classified under ischemic and septic etiologies [19–21].
Rat renal ischemia–reperfusion (I/R) model Operative procedures were performed in rats under general anesthesia induced by the combination of ketamine, 80 mg/kg, and xylazine, 10 mg/kg, administered intraperitoneally. Renal ischemia was induced by unilateral left renal artery clamping, using a small nontraumatic vascular clamp, for 45 min [12]. Reperfusion was initiated by removal of the clamp. The period of renal reperfusion was 24 h. Preliminary experiments revealed no difference between the I/R rat contralateral kidney and sham-operated normal rat kidneys with regard to cell death of renal tubular epithelium after 24 h. Thus, in the present study, the contralateral, untreated kidney from the same animal served as the sham-operated control kidney. During the course of reperfusion, animals were injected intravenously with 20 mg/kg of DDC and 4 h later both kidneys were excised, frozen in liquid nitrogen, and stored at −70°C until use. For microscopic analysis and fluorescence imaging, 5-μm-thick cryo-sections of both kidneys were evaluated either by fluorescence microscopy or were stained with hematoxylin and eosin (H&E) for light microscopy analysis of apoptotic and/or necrotic lesion distribution. Furthermore, the presence of apoptotic cell nuclei was confirmed by apoptosis detection kit staining (ApopTag Fluorescein Kit, Intergen Company, Purchase, NY) using the TUNEL (terminal deoxynucleotidyl transferase-mediated uridine triphosphate nick end labeling) method. Additional ex vivo whole organ fluorescence imaging was carried out on freshly excised kidneys analyzed under a Leica (Leica MZ FL III, Leica, Switzerland) stereomicroscope. DDC was visualized using a 360Ex and >420Em nm (NU) filter set. Emitted fluorescence was collected on a Leica DC 300F digital camera and images were processed and analyzed by Leica QWin Lite software. For quantitative analysis of the extent of renal DDC accumulation, previously frozen kidneys were thawed, weighed, and homogenized in buffer containing 50 mM Tris-HCl and 0.005% Triton X-100, pH 7.4 (1:7 w/v), using Heidolph RZR 2020 homogenizer (Heidolph Instruments GmbH & Co. KG, Schwabach, Germany). Solubilized tissue extract samples were centrifuged twice at 13,800 rpm (20 min, 4°C) and the supernatant fractions were analyzed using a microplate reader (GENious Fl Reader, TECAN, Grödig, Austria) equipped with optical filters for fluorescence measurements. All samples were pipetted in triplicate onto black Ritter flat bottom microplates and read at 360 nm Ex and 535 nm Em. For the calibration curve, a dilution series of DDC were prepared in the same Tris-Triton buffer, supplemented with 0.05% kidney extract of control vehicle-injected animals. The same control animal samples were also used for background values. In all samples, DDC concentration (μg/g tissue) and the percentage of the injected dose (% ID/g) were calculated.Additional biodistribution studies were carried out to assess the spread of radiolabeled DDC into different organs. Twenty-four hours after unilateral renal ischemia/reperfusion, rats were intravenously injected with 5–20 μg/kg of the tritiated form of DDC (3H-DDC) [111–370 kBq (3–10 μCi)/animal]. Four hours later, both kidneys, liver, gut, and blood samples were collected, weighed, and submitted to lyses by SOLVABLE reagent (GNE9100, Packard Bioscience), according to the manufacturer’s instructions, and sample radioactivity was determined using a beta counter (TRI-CARB 2100TR, liquid scintillation analyzer, Packard Bioscience). Both tissue uptake (% ID/g) and the ratios of the radioactivity of the ischemic kidney (DPM/g) to that of the contralateral kidney and the other non-target tissues were calculated.
Rat model of radiocontrast-induced distal tubular necrosis (DTN) The selectivity of DDC uptake in injured renal tissues was evaluated in this rat model, characterized by selective medullary hypoxic tubular damage. Nephropathy was induced by the combined administration of indomethacin (Sigma Chemical Co.), 10 mg/kg, i.v., N-ω-nitro-L-arginine methyl ester (L-NAME, Sigma Chemical Co.), 10 mg/kg, i.v., and the radiocontrast agent sodium iothalamate 80% (Angio-Conray, Mallinckrodt Inc), 6 ml/kg, i.a., as previously described [22]. The study was performed on six rats, with an additional two rats injected with vehicles serving as control (these two control animals studied are representative of at least 29 control rats tested in our previous experiments). Twenty-four hours after insult, animals were intravenously injected with 20 mg/kg DDC and 2 h later they were sacrificed. The left kidney was removed and snap-frozen for fluorescence analysis (and for H&E and TUNEL staining, as detailed above), whereas the right kidney, used for morphological evaluation, was in vivo fixed with glutaraldehyde perfusion through the abdominal aorta, as described elsewhere [22, 23]. Perfusion-fixed kidneys were immersed in buffered 2% OsO4, dehydrated, and embedded in an Araldite-EM bed 812 mixture. Large sections were cut perpendicular to the renal capsule, containing cortex and medulla, including fornix and papillae. One-micrometer sections were stained with methylene blue and analyzed in a blinded fashion (i.e., without knowing the fluorescence results) for morphological alterations. As previously detailed [23], tubular necrosis was determined separately for S3 proximal tubules in the outer stripe and medullary rays, and for medullary thick ascending limbs (mTALs) in the outer, mid, and inner zones of the inner stripe of the outer medulla. The extent of damage was expressed as the percentage of necrotic tubules out of total tubules counted. Papillary necrosis was semiquantitatively assessed using a score of 0–3.
Sepsis model in the mouse The sensitivity of cell death recognition by DDC was tested in the cecal ligature and perforation (CLP) model in mice, where rare, focal tubular cell damage is hardly detected by regular morphological evaluation [24, 25]. The cecum of six anesthetized mice was isolated, ligated distal to the ileocecal valve, and punctured twice with a 26-gauge needle. An additional four sham-treated animals were laparotomized, but the cecum was neither ligated nor punctured. Twenty-four hours later, mice were injected i.v. with DDC, and 2 h afterwards, kidneys were removed and subjected to fluorescence microscopy analysis as detailed above.
Statistical analysis
Data reported are mean±s.e.m. Student’s t test was used to assess the significance of differences between two groups. Significance was accepted at p<0.05
Results
Binding of ApoSense to apoptotic cells in vitro
Concomitant confocal imaging of HeLa cells undergoing apoptosis, double stained with DDC and annexin V, revealed that DDC accumulates within the cytoplasm of the apoptotic cell while annexin V is attached to the external apoptotic cell membrane (Fig. 1). Accumulation of ApoSense within apoptotic cells can contribute to amplification of the signal obtained and consequently to the high signal-to-noise ratio. Previous studies have shown that through an interaction with the death receptor CD95 induces apoptosis by formation of a signaling complex at the cell membrane and subsequent caspase-8 and caspase-3 activation [26]. Therefore, the specificity of DDC uptake was tested in apoptotic Jurkat cells that were co-treated with caspase inhibitor and analyzed by flow cytometry. As shown in Fig. 2, in viable, non-apoptotic cells (green line), no uptake of DDC was detected, except in a small population of spontaneously dying cells representing late events. Following 150 min of incubation with anti-Fas, most cells accumulated DDC and created a new peak of cells (red line) with a higher fluorescence intensity (the geometric mean fluorescence intensity value, GMFI, was 120.18) while the peak of late apoptotic cells remained unchanged. In the presence of the caspase inhibitor Z-VAD-FMK, cells lost the ability to bind DDC (black line), while their viability remained preserved, suggesting that accumulation of DDC compound is dependent on the apoptotic process itself. Upon addition of the caspase inhibitor, a marked shift towards a lower fluorescence intensity was noted, and the final intensity values were almost the same as the control cell values (GMFI 46.38 and 46.02, respectively), suggesting maximal inhibition. Fig. 1Concomitant confocal imaging of HeLa cell undergoing apoptosis by DDC (green fluorescence) and annexin V (red fluorescence). DDC accumulates within the cytoplasm of the apoptotic cell while annexin V is attached to the external membraneFig. 2Binding of DDC to apoptotic cells is caspase dependent. Jurkat cells were treated with CD95 in the absence (red line) or presence (black line) of the pan caspase inhibitor z-VAD-fmk for 150 min. Following incubation, cells were centrifuged and incubated in the presence of DDC for 40 min before FACS analysis. This histogram charts the number of cells counted (y-axis) and the fluorescence intensity (x-axis). The green line represents the control non-apoptotic sample
ApoSense uptake into ischemia/reperfusion (I/R) injured kidneys
Evaluation of DDC accumulation into injured tubular cells of an ischemic rat kidney was performed on the basis of its intrinsic fluorescent properties. Either ex vivo whole kidney fluorescent imaging or cryo-section fluorescent analysis was employed. As seen in Fig. 3, DDC accumulated in the injured (a) but not in the contralateral intact kidney (b), thereby exhibiting substantial capability for imaging apoptosis and/or necrosis in vivo. Microscopic analysis of corresponding kidney sections revealed intense cytoplasmatic staining of tubular epithelial cells (Fig. 3c,d) located at the corticomedullary junction. No uptake of DDC into viable renal parenchyma was detected. The extent of DDC accumulation in these cells was quantitatively determined in homogenized whole kidney extracts from five rats, using microplate reader fluorescence measurement. As shown in Fig. 3e, the average DDC uptake into the ischemic kidney (expressed as %ID/g) was 6.73±1.34 fold higher (p=0.0076) than that in the intact control kidney (0.41±0.11 and 0.06±0.02, respectively). H&E-stained histological sections of kidneys from I/R-treated rats revealed multiple apoptotic as well as necrotic cells situated along the tubule epithelium or in the tubule lumen (Fig. 4). A spectrum of changes characteristic of tubular cell apoptosis, including shrinkage of renal tubular cells, cytoplasmic acidophilia, chromatin dense condensation and margination, nuclear fragmentation, and pyknosis, is shown in Fig. 4a, left. In addition, as previously described [27–30], a large percentage of tubules in this region showed substantial tubular dilatation, epithelial necrosis, dissolving of tubular cells, luminal necrotic debris, and cast formation (Fig. 4b, left). The presence of apoptotic/necrotic cells was also defined by intensive green fluorescence of TUNEL-positive nuclei (Fig. 4c, left). As shown in Fig. 4a–c, right panel, the accumulation of DDC within the damaged areas in ischemic kidneys corresponds completely, even at the single cell level, to the injured areas detected by H&E and TUNEL staining. However, it seems that in some areas, accumulation of DDC in apoptotic cell cytoplasm occurred earlier than DNA destruction and thereby cells were not stained by TUNEL but merely by DDC (Fig. 4c, arrow). These results may indicate that DDC has the ability to identify early apoptotic cells and that use of TUNEL alone may not reveal the full spectrum of cell death stages. Fig. 3DDC uptake into renal damaged areas. a,b Ex vivo images of the left ischemic (a) and the right sham-operated (b) kidney derived from a single I/R rat (×7). Whole organs were viewed under a stereomicroscope using a NU filter set without light flux, 4 h after DDC administration. c,d Histological sections of kidneys (c, ×100; d, ×600) revealing intense uptake along the tubule epithelium or in the tubule lumen. e A quantitative estimate of DDC uptake into I/R ischemic and intact (sham) kidney from five rats, normalized to total injection dose for each gram of tissue. Values are means±s.e.m.Fig. 4Morphological evidence for renal tubular cell apoptosis/necrosis following ischemia–reflow. a Tubular cells (H&E staining) are seen with changes typical of apoptosis (left). In an adjacent 5-μm section (right), these cells take up DDC. ×1,000. b Necrotic tubular cells in tubular lumina (H&E staining; left) and corresponding DDC fluorescence of the same injured cells (right). ×400. c Co-localization of DCC fluorescence (right) with TUNEL-positive nuclear staining (left). Some cells are likely in the early stages of apoptosis and are TUNEL negative, but take up DDC (arrow). ×200
Biodistribution studies of 3H-DDC were performed in seven additional rats following the I/R procedure. The accumulation of radioactivity in the blood, gut, liver and kidney at 4 h after intravenous administration is shown in Fig. 5. Uptake in the ischemic kidney was significantly higher than that in the intact contralateral one(p<0.05) and also higher than liver, gut and blood uptake values (p<0.01). The average uptake values at 4 h, rendered as % ID/g, were 1.20±0.13 for the ischemic kidney, 0.58±0.12 for the intact kidney, 0.41±0.07 for the liver , 0.42±0.09 for the gut, and 0.28±0.01 for blood. The corresponding mean ischemic kidney-to-tissue ratios were 2.1±0.5, 2.9±0.5, 2.8±0.7, and 4.3±0.6, respectively. These results indicate that at 4 h following administration, DDC remained accumulated in the target organ, while being cleared from non-target ones. Fig. 5Biodistribution of radiolabeled 3H-DDC after renal I/R treatment. Uptake of 3H-DDC (% injected dose per gram corrected for background) in target and non-target tissues, determined 18–24 h after ischemia and 4 h after 3H-DDC administration, is shown. Data are presented as mean±s.e.m., n=7. Asterisks denote significantly higher accumulation of 3H-DDC in ischemic kidney compared with other organs (tissues): *p<0.05, **p<0.01
Radiocontrast-induced distal tubular necrosis
Heyman et al. [23] reported previously that animals subjected to the radiocontrast model of DTN developed varying degrees of renal dysfunction and structural damage, but the presence, distribution, and extent of tubular injury on an individual basis could not be predicted from the magnitude of kidney dysfunction. In the present study, as well as in previously published studies [22, 23, 31], renal morphology analysis disclosed a wide-ranging extent of medullary (namely mTALs and S3 segments in the outer medulla and medullary rays) and papillary damage, with a poor overall correlation between structural damage and functional deterioration (data not shown). By contrast, as shown in Figs. 6 and 7, DDC distribution pattern and degree of deposition closely mirrored the morphological findings in the contralateral perfused kidney, suggesting that ApoSense uptake may reflect the level and degree of renal damage more closely than do functional tests. Results from both fluorescence and morphological analysis disclosed the characteristic striped pattern of injury (Fig. 6a,b), where tubules adjacent to the vasa recta are preserved while those distant from vascular bundles are injured (Fig. 7a). Homing of DDC was primarily restricted to injured regions within the outer medulla (Fig. 6c). No DDC uptake at a specified region in the absence of morphological damage was seen, as demonstrated in Fig. 7b, where necrosis of the papillary tip in one of the kidneys was clearly morphologically demarcated and DDC deposition was restricted to precisely the same injured area. Uptake occurred preferentially in apoptotic/necrotic lesions, as confirmed by co-localization with TUNEL staining (Fig. 6d). Thus, DDC was found to be a specific molecular marker for tubular injury in various segments of the kidney. Fig. 6Renal macroscopic and microscopic findings in rats subjected to the ARF protocol (indomethacin, L-NAME and iothalamate) and subsequently injected with DDC. a Macroscopic view of the characteristic striped pattern of severe hypoxic outer medullary damage in an in vivo fixed kidney. b The in vivo microscopic distribution of DDC in the contralateral kidney correlates closely with the distribution of the striped pattern of outer medullary necrosis in the ipsilateral one (×30). c Details (×100) of the cross-section, showing fluorescent imaging of DDC uptake in injured areas of both the inner strip and the forniceal region of the outer medulla. Examination of contiguous 5-μm sections revealed that the accumulation of DDC in damaged areas (d, upper panel) matched the infarct areas stained by TUNEL (d, lower panel) (×200). OM outer medulla,IM inner medulla, SP secondary pyramidFig. 7Characteristic morphological findings in rats subjected to the ARF protocol and injected with DDC. Cryo-sections from the left kidney were used for DDC fluorescence analysis, whereas the in vivo fixed right kidney was used for light microscopy morphological examination of methylene blue-stained slides. Analyses of both kidneys were performed in a blinded fashion. An example is shown of the striped pattern reflecting the damage gradient, where tubules adjacent to the vasa recta (VR) are preserved (red stars) while those distant from vasa recta are injured (black stars) (a, left panel, ×100). Note the excellent co-localization of morphologically damaged tubules with the DDC fluorescence (a, right panel, ×100) in the contralateral kidney. DDC fluorescence was selectively identified in all morphologically injured regions, including papillary tip structures (b: left panel ×40, right panel ×100)
Cecal ligature/perforation-induced sepsis
DDC was evaluated as a potential in vivo marker for renal tubular cell apoptosis in another animal model of ATN, in which sepsis-associated renal dysfunction was induced by cecal ligation and puncture. Morphological alterations of the kidney due to sepsis can hardly be identified using routine histological techniques [32, 33], and experimental data regarding the apoptotic cells of kidneys affected by sepsis are therefore almost non-existent. Indeed, in all septic mice we found only rare focal renal tubular cell injury, seen either in the outer medulla or in the cortex (Fig. 8a). As seen in Fig. 8b, focal renal tubular cell injury is apparently identified by DDC fluorescence, but is not clearly traced using H&E histopathological analysis; this demonstrates the high sensitivity of DDC in the detection of such renal damage. TUNEL staining confirmed the presumption that renal cells stained by DDC are indeed dying cells (Fig. 8c). Fig. 8Focal uptake of DDC in kidneys of six mice following sepsis caused by cecal ligature and perforation (CLP). Apoptotic cells, imaged by DDC, were shown either in the outer medulla (a, left) or in the cortex (a, right) of individual mice (×100). Most apoptotic cells appeared to be tubular epithelial cells (b, left, arrows). An evident correlation was demonstrated between DDC fluorescence (b, right and c, right) and apoptotic cells detected on H&E (b, left) or TUNEL (c, left) stained slides (×200)
Discussion
As a result of recent progress in medicine, biology, and physics, the field of diagnostic imaging is shifting from conventional anatomical imaging to the sphere of functional and molecular imaging, with the aim of imaging biological processes related to both health and disease. The implementation of the new ApoSense technology for clinical imaging of apoptosis would represent a major addition to this emerging field, allowing non-invasive imaging of cell death processes in vivo as a means to both the diagnosis of disease and the monitoring of treatment efficacy in a broad spectrum of disease states.
In the present study, we have reported the successful application of ApoSense technology as a diagnostic imaging tool for the detection of apoptotic and/or necrotic cell death. ApoSense technology may be particularly useful in ARF, where current clinical and physiological assessment and complementary imaging tests are insufficient to discriminate between true renal parenchymal damage and prerenal azotemia (in which kidney filtration function is drastically reduced even though the internal structures of the kidney are intact). An inherent fluorescent property of one of the ApoSense family of compounds, DDC, was utilized for detection of tubular cell death in three animal models of ATN. The results obtained are in complete agreement with in vitro findings of DDC-specific binding to apoptotic cells (Figs. 1, 2), and thereby strongly suggest the ability of DDC to image renal tubular cell apoptosis in vivo.
The three in vivo experimental models of ATN used in the present study complement each other regarding the applicability of ApoSense technology in the detection of ATN. The results obtained using the I/R model showed the qualitatively remarkable capability of ApoSense in targeting of injured tissues and ex vivo imaging of apoptosis (Fig. 3). Additionally, the model provides quantitative analysis of the extent of renal tubular cell damage based on the extent of DDC accumulation in these cells. It was shown that the uptake of DDC in the ischemic kidney was 6.73-fold higher than in the non-ischemic one. Moreover, the specificity of targeting of the injury by ApoSense was demonstrated by its localization to regions of apoptotic/necrotic cell death, detected by H&E and TUNEL staining (Fig. 4). Furthermore, it was observed that some TUNEL-negative cells with a normal overall nuclear morphology were distinctively stained by ApoSense (Fig. 4c), conceivably representing cells in the initial phases of apoptosis. These results possibly indicate that ApoSense technology can provide a tool which will directly assess for early stages of programmed cell death in the ischemic kidney, before membrane vesicle formation and DNA degradation, particularly as measured by the TUNEL method [34].
The radiocontrast model of distal tubular necrosis provides a good example of specific targeting by ApoSense. Indeed, the presence of DDC was restricted to regions (medullary outer and inner stripe; papillary tip) that corresponded closely to the injured zones in the contralateral, perfusion-fixed kidney (Figs. 6, 7). Methylene blue-stained thin sections from those in vivo fixed kidneys enabled precise evaluation of the renal morphology and accurate identification of tubular cell types at various stages of evolving structural damage. The complete correlation between DDC fluorescence and histological evidence of damaged areas, mainly made up of apoptotic and necrotic cells, is indicative of the specificity of DDC uptake. Thus, while the degree of renal dysfunction served as an inaccurate indicator of tubular injury [4, 5], ApoSense uptake closely predicted the existence of ATN, with an absolute specificity for involved tubular segments.
Our findings derived from the mouse sepsis model further illustrated the sensitivity of ApoSense in the detection of rare focal tubular cell injury that is not well defined by routine H&E staining (Fig. 8). It has to be reiterated that data regarding the morphology of sepsis-related ATN are nearly non-existent, mainly because such rare, focal tubular cell damage can hardly be detected by regular morphological evaluation [35, 36].
The inherent fluorescent properties of the ApoSense molecule DDC have been used in the present study to evaluate its specific binding to apoptotic cells. On the other hand, the structure of ApoSense molecules is modular, allowing for versatile attachment of various clinical imaging markers and, therefore, enabling their use as non-invasive imaging agents. In the current study, a 3H-labeled DDC molecule was used for biodistribution and quantitative uptake studies. Nevertheless, autoradiography with radiolabeled DDC was not performed in this series of experiments and this may be the main limitation of the present study. Moreover, additional studies based on preparation of ApoSense as a PET imaging agent (following its conjugation with 18F) are necessary before the introduction of the technology in the form of diagnostic imaging tests. These studies are currently in progress.
Overall, the results presented here are promising and illustrate the high sensitivity and specificity of ApoSense in the assessment of tubular cell damage in multiple experimental templates that may have clinically relevant applications. For instance, ApoSense technology may be implemented for the in vivo assessment of the degree to which renal parenchymal cell apoptosis is contributing to renal failure in sepsis and for the differentiation of evolving ATN from renal hemodynamic alterations; as a result it can enable therapy to be tailored to each patient’s needs. The ability of radiolabeled derivatives of ApoSense to visualize both apoptosis and necrosis, non-invasively and in real-time, may prove useful in reducing the need for routine biopsy. Furthermore, in clinical practice, ApoSense imaging may provide diagnostic information superior to that obtained by means of biopsy, especially as regards the dynamic nature and focal distribution of the disorder; furthermore, it does not suffer from the risks and limitations associated with tissue sampling.
Imaging sciences have grown tremendously during recent decades, and many non-invasive techniques for the detection and monitoring of renal hemodynamics, such as advanced ultrasonography techniques, magnetic resonance imaging, and nuclear and X-ray computed tomography [37–39], have become essential clinical tools. Similarly, non-invasive technologies and imaging probes that would enable the detection of evolving renal parenchymal apoptotic and necrotic injury are critically needed. During the past few years, the best-characterized apoptosis marker, annexin V, a calcium-dependent phospholipid binding protein [40, 41], has been extensively investigated in multiple animal models as well as in clinical trials [42]. However, annexin V has a strong affinity for intact renal cortex, which has been attributed to the intrinsic lipid profile of the kidney, where a high concentration of phosphatidylserine exists [43, 44]. The high accumulation of annexin V in the kidneys precludes its use for the study of apoptosis in these organs or in their immediate vicinity [45, 46]. In addition, the biological rationale for engineering DDC, a small non-peptide molecule with a molecular weight of 700 Da (as compared with the 36-kDa annexin V protein), is that such small molecules could benefit from less immunogenicity and the favorable biodistribution kinetics that small compounds in general exhibit over bigger proteins.
In summary, this study demonstrates that ApoSense, once successfully radiolabeled with an imaging moiety, may provide a sensitive means for real-time diagnosis and/or monitoring of the extent of renal damage in ATN, utilizing selective targeting, binding, uptake, and accumulation within renal tubular apoptotic and/or necrotic cells. Moreover, as a non-invasive method, ApoSense imaging would allow longitudinal studies in a single individual, rendering important information on the optimal timing and dosing of drugs and on the efficacy of therapeutic interventions.

apoptosis imaging
cell death detection
renal disease
renal uptake
small animal imaging


Acute and constitutive increases in central serotonin levels reduce social play behaviour in peri-adolescent rats

Rationale Serotonin is an important modulator of social behaviour. Individual differences in serotonergic signalling are considered to be a marker of personality that is stable throughout lifetime. While a large body of evidence indicates that central serotonin levels are inversely related to aggression and sexual behaviour in adult rats, the relationship between serotonin and social behaviour during peri-adolescence has hardly been explored.
There is a large body of literature that describes the association between serotonin (5-hydroxytryptamine, 5-HT) signalling, social status, aggression and sexual behaviour in humans and animals. For instance, cerebrospinal fluid (CSF) 5-hydroxyindoleacetic acid (5-HIAA; the major metabolite of 5-HT) levels are inversely related to aggression in humans, primates and rodents (Higley and Linnoila 1997; Fairbanks et al. 2001). Selective serotonin reuptake inhibitors (SSRIs), which are well-known anti-depressants, reduce impulsive aggression as well as sexual behaviour in humans (New et al. 2004; Olivier et al. 2006). Furthermore, depending on the social structure of the community, serotonergic drugs are able to interchange the dominant and subordinate status of community members (Edwards and Kravitz 1997; Larson and Summers 2001). It has been proposed that individual differences in 5-HT neurotransmission are an important neural underpinning of personality (Serretti et al. 2006), because central 5-HT levels are relatively stable throughout lifetime (Higley and Linnoila 1997). Studies showing that polymorphisms in 5-HT-related genes are linked to impulsive aggression in humans (Ferrari et al. 2005; Haberstick et al. 2006; Popova 2006) support the idea that the serotonergic modulation of social behaviour is heritable (Higley and Linnoila 1997).
An important form of social behaviour is social play behaviour, which is the earliest form of non-mother-directed social behaviour in young mammals. Social play behaviour consists of behaviours found in adult sexual, affiliative and aggressive encounters (Bolles and Woods 1964; Baenninger 1967; Poole and Fish 1975; Meaney and Stewart 1981). However, during social play, these behaviours are displayed in an exaggerated and/or out-of-context fashion (Poole and Fish 1975). In rats, a bout of social play behaviour starts with one rat soliciting another animal, by attempting to nose or rub the nape of its neck. If the animal that is solicited upon rotates to its dorsal surface, ‘pinning’ is the result, i.e. one animal lying with its dorsal surface on the floor with the other animal standing over it. From this position, the supine animal can initiate another bout of play, by trying to gain access to the other animal’s neck. Thus, during social play, pinning, which is considered to be the most obvious posture in social play behaviour in rats, is not an endpoint but rather functions as a releaser of a prolonged play bout (Poole and Fish 1975; Pellis and Pellis 1987; Pellis 1988). The animal that is pounced upon can also respond by evading or by turning around to face the other animal. In the latter situation, a brief period of boxing/wrestling may follow, in which the animals try to push each other away. If the solicited animal evades, the other animal may start to chase it, thus making another attempt to launch a play bout (see Vanderschuren et al. 1995a, for a detailed analysis of the temporal structure of social play behaviour in rats). It is thought that social play sub-serves the facilitation of social and cognitive development because play deprivation, i.e. social isolation during 2 weeks in peri-adolescence when social play is most abundant (i.e. from postnatal day 21 until postnatal day 35), leads to behavioural disturbances, most prominently in the social domain (e.g. Hol et al. 1999; Van den Berg et al. 1999). These behavioural changes are more selective than those of isolation rearing, which entails continuous isolation from weaning throughout further lifetime. This not only affects social behaviour (Ferdman et al. 2007) but also induces behavioural disturbances related to exploratory behaviour (Heidbreder et al. 2000), cognition (Heidbreder et al. 2000; Dalley et al. 2002; Bianchi et al. 2006) and reward sensitivity (Howes et al. 2000), suggesting that social play plays a rather selective role in the development of social skills.
There is evidence indicating that 5-HT modulates social play in non-human primates. For instance, low CSF 5-HIAA levels are associated with increased solitary play (Maestripieri et al. 2006; Higley and Linnoila 1997), although these findings are difficult to interpret in terms of central 5-HT levels. The neurobiology of social play has been widely investigated in rats (Vanderschuren et al. 1997; Siviy 1998), but surprisingly, little attention has been paid to the role of the serotonergic system in rat social play itself. While treatment with para-chloro-phenylalanine or a low tryptophan diet, resulting in a decrease in central 5-HT levels, did not affect social play, fenfluramine and the SSRI fluoxetine, agents that increase extra-neuronal 5-HT levels, inhibited play (Panksepp et al. 1987; Knutson et al. 1996). Further, the 5-HT receptor agonist quipazine reduced social play (Normansell and Panksepp 1985). These data suggest that there is an inverse relationship between 5-HT signalling and social play. Furthermore, the findings that 5-HT modulates social behaviour in peri-adolescent animals indicates that 5-HT affects social behaviour throughout development, consistent with the proposed trait-like relationship between 5-HT and social behaviour.
The serotonin transporter (SERT) critically regulates extracellular 5-HT levels by its re-uptake into pre-synaptic terminals (Lesch et al. 1996), and SERT knockout models would therefore be very useful to test the hypothesis that central 5-HT levels and social play behaviour are inversely related in a trait-like manner. SERT knockout mice are available (e.g. Bengel et al. 1998), which display reduced aggression (Holmes et al. 2002a) and social interaction (Kalueff et al. 2007). However, because social play behaviour in mice is much less developed than in rats (Poole and Fish 1975; Pellis and Pasztor 1999), mice are not the preferred rodent species to study age-specific patterns of social behaviour during development. As social play behaviour in rats has been well described (Bolles and Woods 1964; Baenninger 1967; Poole and Fish 1975; Meaney and Stewart 1981; Pellis and Pellis 1987; Pellis 1988; Vanderschuren et al. 1997), we measured social play in the recently generated SERT knockout rat (Smits et al. 2006), which displays a ninefold increase in extra-neuronal 5-HT levels (Homberg et al. 2007). In line with the findings that increases in 5-HT levels reduce aggressive and sexual behaviour, we have observed that both aggressive and sexual behaviour (Homberg et al., unpublished observations) are reduced in the SERT knockout rat. The constitutive absence of the SERT in mice causes changes in the functioning of 5-HT receptors (Fabre et al. 2000; Bouali et al. 2003), so that changes in behaviour may be the result of adaptations that have occurred during development. Therefore, we also investigated social play behaviour in wild-type rats after acute treatment with compounds that increase central 5-HT levels, i.e. the SERT-blocker fluoxetine and 3,4-methylenedioxymethamphetamine (MDMA or “ecstasy”), which causes 5-HT release by reversing 5-HT transport.
Materials and methods
Subjects
All experiments were conducted with the approval of the animal ethics committees of the VU Medical Center, Amsterdam, The Netherlands, and the University Medical Center Utrecht, The Netherlands.
Both commercially supplied and SERT knockout rats were used in this study. Male Wistar rats (Harlan, Horst, the Netherlands) arrived at the age of 3 weeks in the animal facility. SERT knockout rats, which have been generated by ethylnitrosourea-induced mutagenesis in a Wistar background (for detailed description, see Smits et al. 2006), were bred by crosses between outcrossed (six generations) SERT+/− rats. At the age of 2 weeks, ear cuts were taken under anaesthesia and used for genotyping. At the age of 3 weeks, male SERT−/− and SERT+/+ rats (littermates served as controls) were transported from the breeding room to the experimental room. The animals were socially housed (four rats per type IV macrolon cage) under a normal day/light cycle (lights off at 7 p.m.) at controlled room temperature (21 ± 2°C) and relative humidity of 60 ± 15%. Food and water were available ad libitum. Three days after arrival, the rats were weighed and handled daily until testing.
Behavioural procedure
Testing was performed as described previously (Vanderschuren et al. 1995b). Rats, aged 28–35 days, were tested in an acrylic plastic cage (40 × 40 × 60 cm [l × w × h]) with approximately 2 cm of wood shavings covering the floor. The test cage was illuminated by a 25-W red light bulb mounted 60 cm above the test cage. Background noise, produced by a radio, was used to minimize the influence of extraneous sounds.
Two days preceding the test, the animals were habituated to the test cage during 10 min. The animals of a test pair did not differ more than 10 g in body weight and had no previous common social experience. On the test day, test pairs were isolated for 3.5 h before the test to induce a half-maximal increase in the amount of social play behaviour (Niesink and Van Ree 1989). Thirty minutes before the test, pairs of animals in group 1 were treated with 0 or 1 mg/kg fluoxetine (subcutaneous [s.c.]), animals in group 2 were pre-treated with 0 or 10 mg/kg fluoxetine and group 3 received 0, 0.5, 2 or 5 mg/kg MDMA (s.c.). Pairs of similarly treated rats were tested for 15 min in the test cage and the test order was randomized across treatments. Behaviour of the animals was recorded on video tape, and analysis from the video tape recordings was performed afterwards. Coding of the drug solutions ensured that both during experimentation and analysis, the experimenter was unaware of the treatment of the animals. Using Observer 4.0 (Noldus Information Technology B.V., Wageningen, The Netherlands), frequencies and durations of the following behaviours was scored—pinning: one of the animals lying with its dorsal surface on the floor of the test cage with the other animal standing over it; pouncing: play soliciting by nosing the partner’s nape; boxing/wrestling: facing each other in vertical position and struggling using the forepaws; following/chasing: moving in the direction of or pursuing the test partner, who moves away; social grooming and social exploration: sniffing or licking any body part of the test partner. Behaviour was assessed per pair of animals. Animals were used only once.
Drugs
Fluoxetine was purchased from Sigma-Aldrich (Schnelldorf, Germany), and MDMA was purchased from O.P.G. (Utrecht, The Netherlands). Both drugs were freshly dissolved in saline on the day of the experiment and injected s.c. in the flank in a volume of 2 ml/kg.
Statistical analyses
Data were analysed using one-way analysis of variance (ANOVA), followed by Student–Newman–Keuls post-hoc tests where appropriate. The level of probability for statistically significant effects was set at p < 0.05 (n.s. = non-significant).
Results
SERT−/− pairs of rats, as compared to SERT+/+ pairs, showed a strong reduction in pinning frequency (Fig. 1a; F(2, 13) = 5.78, p < 0.05), pouncing frequency (F(2, 13) = 7.08, p < 0.05) and boxing/wresting frequency (F(2, 11) = 9.91, p < 0.01). The duration of following/chasing was significantly increased in SERT−/− rats (Fig. 1b; F(2, 13) = 6.5, p < 0.05), while social grooming and social exploration were not affected by deletion of the SERT gene (social exploration: F(2, 12) = 0.22, n.s.; social grooming: F(2, 12) = 0.76, n.s.).
Fig. 1Social play behaviour in peri-adolescent male SERT−/− and SERT+/+ rats. The data represent mean ± SEM of the number of pins, pounces and boxing/wresting episodes (a) and mean ± SEM of the duration of social exploration and following/chasing (b) during a 15 min test. Asterisk, p < 0.05 SERT−/− vs SERT+/+
As illustrated in Fig. 2a, 1 mg/kg fluoxetine did not affect pinning (F(2, 13) = 1.33, n.s.), pouncing (F(2, 13) = 2.56, n.s.) and boxing/wrestling (F(2, 13) = 1.75, n.s.). One-way ANOVA did not also reveal effects of 1 mg/kg fluoxetine on social exploration (F(2, 13) = 0.55, n.s.), following/chasing (F(2, 13) = 0.69, n.s.) or social grooming (F(2, 13) = 0.00, n.s.; Fig. 2b). A higher dose of fluoxetine, 10 mg/kg, on the other hand, significantly reduced pinning (F(2, 14) = 15.86, p < 0.005), pouncing (F(2, 14) = 36.18, p < 0.0001), boxing/wrestling (F(2, 14) = 23.82, p < 0.0005; Fig. 2c) and following/chasing (F(2, 14) = 20.17, p < 0.001) but not social exploration (F(2, 14) = 3.03, n.s.) and social grooming (F(2, 14) = 0.03, n.s.; Fig. 2d).
Fig. 2Effect of fluoxetine on social play behaviour in peri-adolescent male wild-type rats. Thirty minutes before the 15-min test, the animals were treated with 1 (a, b) or 10 mg/kg (c, d) fluoxetine (s.c.). The data represent mean ± SEM of the number of pins, pounces and boxing/wresting episodes (a, c) and mean ± SEM of the duration of social exploration, following/chasing and social grooming (b, d). Asterisk, p < 0.05 vs saline
MDMA was tested in three different doses: 0.5, 2.0 and 5.0 mg/kg. One-way ANOVA indicated that MDMA treatment dose-dependently suppressed pinning (Fig. 3a; F(2, 30) = 28.8, p < 0.0001, post-hoc testing: all MDMA groups vs saline p < 0.05), pouncing (F(2, 29) = 31.77, p < 0.0001, post-hoc testing: all MDMA groups vs saline p < 0.05) and boxing (F(2, 29) = 22.07, p < 0.0001, post-hoc testing: all MDMA groups vs saline p < 0.05). In addition, MDMA decreased following/chasing (Fig. 3b; F(2, 29) = 8.81, p < 0.0005, post-hoc testing: all MDMA groups vs saline p < 0.05). Social exploration (F(2, 30) = 6.41, p < 0.005, post-hoc testing: 2 and 5 mg/kg MDMA vs saline p < 0.05) and social grooming (F(2, 28) = 24.89, p < 0.0001, post-hoc testing: 2 and 5 mg/kg MDMA groups vs saline p < 0.05; Fig. 3b) were also attenuated by MDMA treatment.
Fig. 3Effect of MDMA on social play behaviour in peri-adolescent male wild-type rats. Thirty minutes before the 15-min test, the animals were treated with 0, 0.5, 2 or 5 mg/kg MDMA (s.c.). The data represent mean ± SEM of the number of pins, pounces and boxing/wresting episodes (a) and mean ± SEM of the duration of social exploration, following/chasing and social grooming (b). Asterisk, p < 0.05 vs saline; double cross, p < 0.05 vs 0.5 mg/kg MDMA
Discussion
In this study, we show that increasing extra-neuronal 5-HT levels, either constitutively by deletion of the SERT gene or transiently by pharmacological manipulation (i.e. treatment with fluoxetine or MDMA), decreases social play behaviour in peri-adolescent rats. These effects were specific for playful social behaviours, i.e. pinning, pouncing and boxing/wrestling. Mixed effects were observed for following/chasing, social grooming and social exploration. The similarities between the genetic and pharmacological manipulations of the serotonergic system suggest that reduced social play behaviour in SERT−/− rats is the result of increased extracellular 5-HT levels (Homberg et al. 2007).
Fluoxetine and MDMA are well known to increase central 5-HT levels, but they affect dopaminergic neurotransmission as well, raising the possibility that reduced social play is due to an effect on the dopaminergic system. Fluoxetine, at 10 mg/kg as used in the present study, increases dopamine release in the frontal cortex (Tanda et al. 1994). MDMA is a potent releaser of 5-HT, but it releases dopamine as well, albeit at higher concentrations (for reviews, see White et al. 1996; Green et al. 2003). Although psychostimulant drugs that increase central dopaminergic neurotransmission have been found to suppress social play behaviour (e.g. Beatty et al. 1982; for review, see Vanderschuren et al. 1997), these effects are most likely mediated through non-dopaminergic mechanisms because the selective dopamine reuptake inhibitor GBR-12909 and the dopamine receptor agonist apomorphine did not suppress play (Vanderschuren et al. 2005). Moreover, the pre-synaptic functioning of dopaminergic, as well as noradrenergic neurons, was not altered in SERT−/− rats (Homberg et al. 2007). Therefore, the present findings are most likely the result of altered function of the serotonergic system, rather than the dopaminergic system.
Indirect 5-HT agonists such as MDMA and parachloroamphetamine enhance locomotor activity (Geyer 1996), but the doses required to enhance locomotor activity in peri-adolescent rats are higher than those that suppress play in the present study (see Åberg et al. 2007). Fluoxetine does not alter locomotor activity (Homberg et al. 2004), and locomotor activity does not differ between SERT+/+ and SERT−/− rats under both a novel and habituated conditions (Homberg et al., unpublished observations). In the present study, following/chasing, the social parameter most closely related to locomotor activity, was increased in SERT−/− rats and decreased by fluoxetine and MDMA. Thus, there is no parallel in the effects of fluoxetine, MDMA and SERT knockout on locomotor activity and following/chasing. This indicates that the reduction in social play found in SERT−/− rats and after treatment with fluoxetine and MDMA is not likely to be secondary to effects on locomotor activity.
An alternative explanation of the present findings is that the decrease in social play behaviour is due to increased anxiety. Although the animals were habituated to the test cage and tested under red light conditions, reluctance to contact an unfamiliar conspecific may have interfered with our findings. SERT−/− mice display anxiety-related symptoms and reduced social interaction (Holmes et al. 2002b; Kalueff et al. 2007), MDMA and fluoxetine have anxiogenic effects in a variety of tests (Lin et al. 1999; Morley and McGregor 2000; Drapier et al. 2007) and the SSRI citalopram reduces social interaction (Dekeyne et al. 2000). MDMA has also been reported to decrease social investigation and aggression and to increase non-social exploration, which was interpreted as attempts to escape from the test arena, i.e. an anxiogenic-like effect of MDMA (Maldonado and Navarro 2001). On the other hand, pro-social effects of MDMA have also been reported (Morley and McGregor 2000). SERT−/− rats spent more time on following and chasing than SERT+/+ rats. This suggests that while social play was reduced, SERT−/− rats were still interested in their playing partner, which argues against social anxiety as an explanation of reduced play behaviour in SERT−/− rats. Moreover, social exploratory behaviour, which is the parameter used in the social interaction test of anxiety (File and Seth 2003), was not affected in SERT−/− and in fluoxetine-treated rats, whereas MDMA only mildly reduced social exploration at doses that completely eliminated social play. Thus, the observed effects were relatively selective for social play, and increases in extra-neuronal 5-HT levels do not induce a generalized aversion to contact an unfamiliar rat.
Long-term SSRI treatment is associated with increased extra-neuronal 5-HT levels and several compensatory adaptations (Dawson et al. 2002) that are strikingly similar to those seen in SERT−/− rats, like desensitization of pre- and post-synaptic 5-HT1A receptors (Homberg et al., unpublished observations). Chronic SSRI treatment decreases aggressive (Fuller 1997) and sexual behaviour (Olivier et al. 2006) in rats, phenotypes we also observed in SERT−/− rats (Homberg et al., unpublished observations). Because acute fluoxetine and MDMA treatment cause an immediate increase in extra-neuronal 5-HT levels without compensatory adaptations, it is likely that reduced social play in SERT−/− rats is due to the increased extra-neuronal 5-HT levels, rather than compensatory adaptations. Ansorge et al. (2004) showed that early chronic SSRI treatment in wild-type mice induces anxiety- and depression-like phenotypes that resembles those of SERT−/− mice. It may follow that SERT−/− rats behave as being chronically treated with SSRIs, but given that acute and chronic SSRI treatment induce opposite effects (Silva and Brandao 2000) and that the effects of anti-depressants on the immature developing brain may differ as opposed to the effects on the mature brain of adults (Ansorge et al. 2004; Taravosh-Lahn et al. 2006), further research is required to support this suggestion. Anti-depressants are frequently prescribed to children with major depressive disorder and anxiety (Cheung et al. 2006), while there is only sparse knowledge of the effects of SSRIs on the developing brain (Spear and Brake 1983; Ansorge et al. 2004). Because social behaviour is reduced in peri-adolescent and adult SERT−/− rats, it is likely that changes in 5-HT homeostasis found at adulthood in SERT−/− rats (Homberg et al. 2007) reflect the situation in the peri-adolescent SERT−/− brain. We thus propose that insight into the function of the serotonergic system in the SERT−/− (peri-adolescent) brain may be helpful to gain insight into the effects of SSRIs on the developing brain.
5-HT is considered to be an important marker of personality traits such as harm avoidance (Gerra et al. 2000), social dominance (Edwards and Kravitz 1997; Larson and Summers 2001), social attachment (Beech and Mitchell 2005) and social impulsivity (Fairbanks et al. 2001), which are relatively stable within individuals. These traits may be the result of changes in SERT function because a polymorphism in the SERT gene of humans and non-human primates, which decreases SERT expression and function (Lesch et al. 1996), has quite consistently been associated with childhood (Beitchman et al. 2006; Haberstick et al. 2006) and adult aggression (Sakai et al. 2006) and childhood (Nobile et al. 2004) and adult depression (Caspi et al. 2003). Furthermore, this polymorphism has been associated with reduced free play behaviour in monkeys (Bethea et al. 2004). Anti-social behaviour in children has been suggested to be related to developmental abnormalities in the serotonergic system, and altered social behaviour during childhood likely extends into adulthood (Van Goozen and Fairchild 2006). Thus, the observations that SERT−/− rats display reduced social play behaviour, reduced aggression and reduced sexual behaviour is likely to be the result of constitutive disruptions in 5-HT function. On the other hand, because social play sub-serves the development of an adequate social repertoire (Vanderschuren et al. 1997; Hol et al. 1999; Van den Berg et al. 1999), the reduction in social play in SERT−/− rats could also itself cause changes in adult social behaviour, independent of 5-HT function. Whichever possibility holds true, we think that because of their lifelong altered 5-HT homeostasis, the SERT knockout rat model may contribute to our understanding of the role of 5-HT during the development of social behaviour.

serotonin
social play
peri-adolescence
fluoxetine
knockout rat
mdma


Harnessing hypoxic adaptation to prevent, treat, and repair stroke

The brain demands oxygen and glucose to fulfill its roles as the master regulator of body functions as diverse as bladder control and creative thinking. Chemical and electrical transmission in the nervous system is rapidly disrupted in stroke as a result of hypoxia and hypoglycemia. Despite being highly evolved in its architecture, the human brain appears to utilize phylogenetically conserved homeostatic strategies to combat hypoxia and ischemia. Specifically, several converging lines of inquiry have demonstrated that the transcription factor hypoxia-inducible factor-1 (HIF1-1) mediates the activation of a large cassette of genes involved in adaptation to hypoxia in surviving neurons after stroke. Accordingly, pharmacological or molecular approaches that engage hypoxic adaptation at the point of one of its sensors (e.g., inhibition of HIF prolyl 4 hydroxylases) leads to profound sparing of brain tissue and enhanced recovery of function. In this review, we discuss the potential mechanisms that could subserve protective and restorative effects of augmenting hypoxic adaptation in the brain. The strategy appears to involve HIF-dependent and HIF-independent pathways and more than 70 genes and proteins activated transcriptionally and post-transcriptionally that can act at cellular, local, and system levels to compensate for oxygen insufficiency. The breadth and depth of this homeostatic program offers a hopeful alternative to the current pessimism towards stroke therapeutics.
Stroke is defined as injury to the brain accruing from a vascular etiology. Strikingly, it has emerged as the third leading cause of death and the leading cause of disability in the USA. Accordingly, the estimated financial costs of stroke are more than 50 billion dollars a year in the USA alone. These financial costs do not begin to tell the story of the personal suffering that amasses from the silent epidemic of stroke disability—over 5 million Americans face the challenges of handicaps from stroke each day. The recognition of stroke as a leading age-associated public health issue has led the government and the pharmaceutical industry to expend enormous resources on developing interventions in the form of drugs that minimize brain damage associated with stroke. Despite their promise, these efforts have been disappointing and have left a nearly indelible sense of frustration on the biomedical research community [1]. In this chapter, we will discuss the pathophysiology of stroke with particular attention to one of its primary mediators, hypoxia, and attempt to revive a sense of optimism and enthusiasm for stroke therapeutics moving forward.
Ischemia is a process in which perfusion to tissue is critically reduced creating a deficit in necessary brain fuels. The brain is highly vulnerable to ischemia because the eloquent functions it is assigned to carry out, in the pluralistic society of organ functions, depend integrally on energy—specifically adenosine triphosphate (ATP). The brain comprises only 2% of body weight, but it utilizes nearly 20% of cardiac output to achieve its supply of essential nutrients including oxygen and glucose. Abnormal central nervous system (CNS) symptoms begin to manifest at 40–50% of normal resting hemispheric cerebral blood flow resulting in slowing of the EEG, attenuation of evoked potentials, and reduction in the membrane potential in individual cortical neurons [2]. The resting membrane potential must be maintained to allow proper neuronal function, including synaptic activity and axonal conduction. Once the neuronal membrane potential begins to dissipate, neuronal function ceases. It is important to note that ATP levels at this point may be normal or only slightly reduced [3]. The reason for this inordinate sensitivity is not entirely clear but is likely related to the Km of neurotransmitter systems for ATP or their exquisite sensitivity to tissue acidosis [4]. Gross deterioration and damage requires even more severe reductions in blood flow to 20–30% of normal resting hemispheric cerebral blood flow (Fig. 1). With this level of ischemia, a deterioration of ionic membrane gradients ensues, and the tissue begins to accumulate hydrogen ions (acidosis) [5]. Changes in ionic fluxes likely accrue directly from a reduction of intracellular ATP, a failure of the Na+/K+ATPase activity, and increases in intracellular Na+ and extracellular K+. The failure of ionic homeostasis reflects loss of activity of multiple transporters that normally maintain the electrochemical gradients necessary for normal neuronal signal transduction [6, 7]. Among the ions deregulated, intracellular calcium appears to play a critical role in further ion dyshomeostasis via the calmodulin-dependent activation of neuronal nitric oxide synthase (nNOS) [8–10]. Increased nNOS activity leads to increase in the ambient levels of neuronal nitric oxide (NO) [11]. Changes in NO coupled with ischemia-associated increases in cytosolic and mitochondrially derived superoxide, combine to form toxic peroxynitrite. Peroxynitrite can trigger cell death pathways via DNA damage, poly(ADP-ribose)polymerase (PARP) activation, liberation of apoptosis-inducing factor (AIF) from the mitochondria, and activation of death signaling pathways leading to apoptosis [12, 13]; alternatively, non-selective cation channels such as TRPM2 and TRPM7 can be activated to ensure sustained calcium overload and death [14]. Extracellular acidosis is also postulated to activate acid sensing ion channels, which also contribute to destruction of the normal ionic environment [15]. As one can appreciate from a partial description of the sequence of events after stroke that occur in the neuron alone, targeting a single molecule in the complex parallel and serial pathways of acute hypoxia-ischemia will not maintain neuronal survival (Fig. 2). It is also unlikely to enhance the ability of energy-thirsty neurons to carry out their sophisticated roles in maintaining posture, movement, sequencing language, or making critical executive decisions. Therefore, how might we move forward? One of the most important advances in the treatment of complex medical problems has been the discovery that multimodal therapies can greatly enhance therapeutic efficiency. Treatment of cancer, HIV infection, and tuberculosis with multimodal therapies yields results that are not obtained with the application of single therapeutic agents.
Fig. 1Neuronal hypoxia leads to calcium overload and production of free radicals. Stroke is associated with a decrease in cerebral blood flow to the brain. The consequent loss of metabolic fuels leads to failure of sodium pumps leading to an intracellular accumulation of sodium and calcium, depolarization, and activation of voltage sensitive and ligand gated [N-methyl-d-aspartate (NMDA)] ion channels. Increased in calcium in microdomains near the NMDA receptor leads to activation of neuronal nitric oxide synthase. Global dysregulation of calcium in the neuron leads to mitochondrial overload and superoxide production. Nitric oxide and superoxide combine to form peroxynitrite. Peroxynitrite can damage DNA leading to PARP activation and consumption of NAD+. It can also activate TRPM2/7 channels leading to further calcium dysregulation. Hypoxia is sensed by decreased activity of HIF prolyl 4 hydroxylases that can lead to activation genetic responses capable of compensating for the sentinel metabolic stress (decreased cerebral blood flow). Decrease HIF PHD activity can also prevent death via HIF-independent pathways. Acidosis and ROS can also combine with HIF regulated prodeath proteins to trigger cell deathFig. 2Contribution of neurons and astrocytes in mediating excitotoxic neuronal death. 1 Loss of ATP in ischemia leads to inhibition of the Na+/K+ATPase and subsequent collapse of normal ionic gradients. 2 In turn, neuronal membrane depolarization activates voltage sensitive Ca2+ channels, which increase intracellular Ca2+ and stimulate vesicular glutamate release. Severe loss of ionic gradients found in certain ischemic regions may also lead to the reversal of 3 neuronal specific and 4 astrocyte specific glutamate transporters, which in the reverse mode act to release glutamate into the extracellular space. 5 Cell swelling in cerebral ischemia, which is mainly localized to astrocytes, likely activates swelling sensitive anion channels, referred to as volume regulated anion channels (VRACs). VRACs, which are permeable to organic osmolytes, contribute to glutamate release predominantly in the ischemic penumbra. 6 Glutamate regulated NMDA receptors (NMDA-R) are activated by (1) extracellular glutamate and (2) release of Mg2+ from its pore after membrane depolarization (in part due to activation of glutamate regulated AMPA receptors, not shown). 7 NMDA-Rs are permeable to Ca2+ and as such, overabundant NMDA-R activation leads to an intracellular Ca2+ overload. This increase in intracellular Ca2+ then contributes to neuronal death via several mechanisms
As stroke does not represent a single homogeneous category of injury, it is also a poor candidate for a single approach to treatment [16]. Challenges involved in promoting recovery from stroke involve reducing the extent of damage that occurs in acute injury. Moreover, many different kinds of damage are found in individuals with stroke ranging from necrotic, apoptotic, or parthanatotic death of neurons [17, 18], demyelination of otherwise functional axons [19], and transection of axons and subsequent loss of critical neuronal populations [20]. Acute injury itself is extremely complex, including waves of cell death, inflammatory responses, edema, and scarring. Indeed, experimental studies have continued to demonstrate that interventions that target single aspects of the complex cascade, including blocking ion gradients, scavenging free radicals, or enhancing growth factors on their own are insufficient to overcome the considerable barriers to protection against hypoxia and ischemia in acute stroke [21].
Instructive data from the experimental paradigm of ischemic preconditioning has pointed the way towards novel strategies that can address the heterogeneity and complexity inherent in stroke pathophysiology [22]. Animals subjected to a non-lethal exposure to hypoxia are found to be more resistant to a host of subsequent lethal stresses, including cerebral ischemia. The mechanism by which a sublethal exposure to hypoxia can render the brain resistant to cerebral ischemia and a host of other insults is a topic of active investigation and debate. However, one point appears irrefutable: The tolerance that develops after a short duration of hypoxia involves not only the activation or inactivation of pre-existing proteins but also de novo gene expression [23, 24]. These transcriptional and post-transcriptional mechanisms reflect a cassette of genes and proteins that work collectively at the cellular, local, and systemic levels to compensate for a discrepancy in oxygen supply and demand. The findings suggest that under conditions where adaptive homeostatic mechanisms are appropriately engaged, damage to the brain can be substantially lessened or even prevented (Fig. 3). Stroke is, almost by definition, a failure of homeostasis. Accordingly, identification of small molecules that augment endogenous adaptive strategies provides a mechanism to tilt the balance away from cell damage and death and toward cell survival and repair (Fig. 3).
Fig. 3Drugs that augment endogenous homeostatic mechanisms will more effectively neutralize the heterogeneity inherent in stroke pathophysiology. As these pathways are already used by the body, their activation can occur with decreased threat of toxicity. The term “homeostasis” was coined by Walter Canon in the early twentieth century. It refers to the innate tendency of organisms to mobilize adaptive responses physiological and pathological perturbations that ultimately return the system to a set point that is consistent with survival. a The experimental paradigm of “ischemic preconditioning” has shown that a short, sublethal exposure to hypoxia, or hypoxia-ischemia induces homeostatic responses that make the organism “immune” or “tolerant” to a lethal ischemic insult. Mechanistic studies have revealed that tolerance is the consequence of activation of pre-existing proteins and de novo gene expression. b According to this model, stroke can be conceptualized as a failure of homeostasis. Consequently, neurons die and the brain is permanently damaged. c By extension, small molecules that engage homeostatic mechanisms designed to alleviate hypoxia/ischemia early or enhance their activation should tip the balance away from cell death and toward survival and repair. Such small molecules are currently being developed and represent a new generation of stroke therapies
Examination of adaptive responses to hypoxia in the central nervous system has affirmed that the family of transcriptional regulators known as the hypoxia-inducible factors are central players [25–27]. HIF-1 was purified and cloned as a result of a search for proteins that regulate the expression of genes involved in hypoxic adaptation, such as erythropoietin, vascular endothelial growth factor, and glycolytic enzymes [27]. HIF is a heterodimeric transcriptional activator composed of an inducible HIF-1α subunit and a constitutively expressed HIF-1β subunit [28, 29]. HIF-1α stability is regulated via the activity of a class of oxygen, 2-oxoglutarate, and iron dependent enzymes known as the HIF prolyl-4 hydroxylases (HIF PHDs, Fig. 4) [30]. As intracellular oxygen levels drop below a critical threshold, these enzymes fail to hydroxylate HIF-1α. As hydroxylation is required for the recruitment of the constitutively active E3 Ubiquitin Ligase, Von Hippel Lindau protein, HIF-1α becomes stabilized. HIF-1α can partner with its constitutively expressed but induced partner HIF-1β and translocate to the nucleus to regulate the expression of a host of genes involved in hypoxic adaptation [31]. Consistent with this model, several groups have shown that HIF-1α immunoreactivity increases in areas of the cortex that become hypoxic due to stroke [25, 32] An unanswered question has been whether HIF is also upregulated in areas connected to but remote from the site of ischemia. Indeed, neurons projecting to an area of infarction are at risk for cell death due to a loss of trophic support from their damaged targets. Moreover, neurons projecting from an area of damage are at risk for cell death due to a loss of trophic excitatory input from their targets. An elegant recent study in non-human primates demonstrated a dramatic increase in neuronal immunoreactivity for HIF-1α and one of its target genes in the infarct and peri-infarct region [33, 34]. Indeed, the neuronal immunoreactivity for HIF-1α increased from less than 5% to nearly 90% in both regions. Interestingly, areas remote to the area of hypoxia and ischemia also experience increased HIF and vascular endothelial growth factor (VEGF) levels but to a quantitatively much smaller extent. Together, the published rodent and primate histochemical studies support the hypothesis that increased HIF protein levels resulting from direct hypoxia and non-hypoxic mediators such as IGF-1 are a marker for surviving and regenerating neurons after ischemia [32].
Fig. 4HIF prolyl 4 hydroxylases sense hypoxia and transduce a critical insufficiency in oxygen in the brain into transcriptional and post-transcriptional signal changes that mediate protection and repair. Hypoxia regulates the activity of HIF PHDs via direct or indirect mechanisms; production of peroxide via reduction in mitochondrial ATP production and electron transport chain (ETC) impairment (1, 2); accumulation of the tricarboxylic cycle (TCA) intermediates succinate and fumarate (3); or direct inhibition of the activity of PHDs due to lack of oxygen (4). Accumulation of hydrogen peroxide, succinate or fumarate can inhibits the activity of PHDs by competing with 2-oxoglutarate or by oxidizing the active site iron (5). Among its numerous downstream effects, inhibition of HIF PHD activity leads to stabilization of HIF-1α. Stabilized HIF-1α dimerizes with HIF-1β in the nucleus and increases gene transcription (6)
Pharmacological and molecular studies have provided additional support for the notion that stabilization of HIF-1 after ischemia is associated with enhanced survival of neurons. Small molecule hypoxia mimics, deferoxamine, and cobalt chloride, were found to stabilize HIF-1α levels, increase DNA binding to a cognate hypoxia response element, and increase the expression of HIF target genes in vitro cultured neurons (Zaman et al. [31]) and in vivo in the intact brain (Fig. 4; [35]). Pretreatment or post-treatment with desferrioxamine or cobalt chloride resulted in reduced cell loss in models of focal or global ischemia in vitro and in vivo [25, 31, 36–39]. Subsequent studies have confirmed that hypoxia, iron chelation, or cobalt chloride appear to confer protective effects on neurons via their ability to inhibit HIF PHDs [35]. Emerging data indicate that inhibition of each of the three of the HIF PHD isoforms (1–3) may lead to cell survival in the nervous system. Of note, inhibition of each isoform may enhance survival via distinct but mutually supportive pathways.
Our studies demonstrate that pharmacological inhibition of PHDs, in vitro, leads to inhibition of oxidative stress-induced death, an established mediator of neural injury and death in stroke [35]. More recent studies indicate that molecular suppression of HIF PHD 1 alone can mimic this effect (Siddiq et al., unpublished observations). Interestingly, while HIF-1, HIF-2, and cAMP response element-binding protein are stabilized by PHD inhibition in neurons or astrocytes, the molecular deletion or inhibition of each of these three transcription factors fails to abrogate the protective effects of PHD inhibition suggesting that other, as yet unidentified, pathways are important in protection (Siddiq et al., unpublished observations). Studies are underway to clarify whether molecular deletion of PHD1 selectively in the CNS confers resistance to stroke in a HIF-independent manner.
In contrast to PHD1, PHD2 appears to be the isoform most important for tagging HIF for degradation. Deletion of PHD2 but not PHD1 or PHD3 results in increased HIF and VEGF protein and consequent angiogenesis in multiple organs including the brain [40]. Underscoring PHD2’s important role in HIF signaling, the expression level of this isoform is significantly higher as compared to its brethren [41]. Ischemia-induced PHD2 inhibition stabilizes HIF and enhances expression of genes that mediate cellular (e.g., glycolytic enzymes), local (vascular endothelial growth factor), and systemic (erythropoietin, Epo) adaptive responses to hypoxia or hypoxia-ischemia [42]. While Epo is best known as a hematopoietic growth factor that can enhance oxygen carrying capacity to tissue, it also has organ-autonomous roles in the nervous system. Epo is produced in astrocytes in response to hypoxia or ischemia and mediates a number of responses critical to stroke prevention and recovery [43, 44]. Epo can inhibit neuronal death due to excitotoxicity or growth factor deprivation [45–49]. It can also stimulate the proliferation of neural progenitors in the germinal zones of the brain and enhance their migration to sites of injury [50, 51]. The concomitant HIF-dependent increase in VEGF expression provides, among other things, the appropriate angiogenic niche for neural progenitors to survive [52]. New neurons could mediate recovery responses via paracrine effects or due to direct participation in functional circuits. A recent study affirmed the requisite role for HIF transcription factors in mediating some of the salutary effects of low molecular weight PHD inhibitors given after cerebral ischemia [53]. Available evidence suggests that these inhibitors are likely targeting PHD2 to induce HIF and its gene targets in the CNS.
Inhibition of the third isoform of PHDs (PHD3) by hypoxia or hypoxia mimetics has also been linked to neuronal survival [54]. Freeman and colleagues first identified PHD3 (then known as SM-20) as a message and protein that is highly upregulated in sympathetic neurons after growth factor deprivation. Subsequent studies have shown that pharmacological or molecular deletion of HIF PHD3 prevents apoptosis associated with trophic loss in neurons and that the protection is HIF independent [55]. As target (post-synaptic) derived trophic factors appear to be lost after stroke and result in cell death remote from the infarct site, HIF PHD3 inhibition may be a rational strategy for maintaining the viability of these neurons in evolving or stable stroke.
From the above discussion, a model begins to emerge whereby inhibition of HIF PHD1, 2, or 3 by hypoxia or consequences of hypoxia can mediate HIF-dependent and HIF-independent compensatory responses via distinct but clearly reinforcing mechanisms. How does one reconcile this model with observations from several laboratories, including our own, that HIF PHD inhibition or HIF activation leads to the upregulation of prodeath, Bcl-2 family proteins such as Puma, Bnip3, and NIX [56]? Moreover, constitutive HIF activation has been associated with potentiation and suppression of death [56]. These findings appear to make perfect sense if one considers apoptosis as an adaptive response to stress. After stroke, oxygen may fall below a critical level rendering the prolyl hydroxylases inactive. Accordingly, HIF is stabilized and it translocates to the nucleus to upregulate genes involved in preventing and executing death. The anthropomorphized cell can be visualized stepping to the edge of a steep cliff in response to a potentially lethal stress (Fig. 5). If adequate compensation for hypoxia occurs, prodeath proteins are not activated and the cell survives as a functional component of a complex neural network—the cell moves away from the cliff to survival promoting, terra firma. By contrast, if oxidative stress, hypoxia, and acidosis dominate, indicating that compensatory mechanisms have failed, then proapoptotic proteins such as BNIP3 undergo a conformational change, insertion into the mitochondrial membrane and activation of permeabilization transition and release of apoptotic effectors [57–59]. In the latter scenario, the cell jumps off the cliff to preserve limiting resources for its neighbors. The model suggests that state changes in the cell (e.g., redox and pH) that follow the initial ischemic insult will determine the fate of the tissue and potential for recovery (Fig. 5). Of note, low molecular weight or peptide inhibitors of the PHDs, antioxidants, and a small interfering RNA to BNIP3 prevent the prodeath effects of HIF (Amino et al., personal communication). These findings suggest that engaging the adaptive response at an upstream point where stress is detected by the cell (the stress sensor) is going to be more effective in stemming cell loss and facilitating repair than at the point of the transcription factor (HIF). It is our hypothesis that engaging the adaptive response at an upstream point in the pathway results in post-transcriptional changes essential for the homeostatic response. These changes in concert with transcriptional changes optimally alleviate the discrepancy between oxygen supply and demand.
Fig. 5Adaptation to hypoxia-cell fate and beyond. Expression of HIF in neurons leads to the constitutive expression of proteins associated with cell death (BNIP3, NIX, and PUMA) and cell survival (VEGF, glycolytic enzymes, Epo, and p21 waf1/cip1). Similar prodeath gene expression is found in neurons exposed to hypoxia or hypoxia mimetics despite the absence of cell death. It appears the oxygen “starved” neurons have stepped to the edge of the cliff. If during the ensuing hours to days the neuron becomes acidotic or oxidized, then prodeath proteins such as BNIP3 undergo a conformational change, insertion into the mitochondrial membrane, release of apoptotic effectors, and death. By contrast, if the survival genes are effective in neutralizing the hypoxic stress (e.g., no acidosis or oxidative stress), then the death genes never get activated. Our studies indicate that antioxidants, short interfering RNAs to BNIP3 or inhibitors of the HIF prolyl 4 hydroxylases tip the balance toward survival (away from the cliff)
The model has some clear predictions. First, low molecular weight global inhibitors of the HIF prolyl 4 hydroxylases will be more effective at preventing injury and repairing damage after stroke than selective isoform inhibitors. These inhibitors will engage HIF-dependent and HIF-independent pathways at cellular, local, and systemic levels and ultimately alleviate the discrepancy in nutrient supply and demand. They can also (via mechanisms that are only beginning to be defined) divert HIF away from its tendencies as a prodeath transcription factor. The ability of single “drugs” to target an oligopoly of proteins (HIF PHD1–3) to affect a concerted program of neuroprotection involving more than 70 genes and larger number of proteins suggests a strategy for overcoming the heterogeneity inherent in stroke pathophysiology in the short term. While a significant amount of work needs to be done to adequately assess the viability of this strategy for human therapeutics, the notion of augmenting endogenous adaptive programs via HIF PHDs to thwart disease continues to gain currency.

stroke
brain
hypoxia
hif
therapeutics
hif prolyl hydroxylase