Physiopathology of radiation-induced neurotoxicity.

Ionizing irradiation for the treatment of malignant brain tumors has associated with it a risk of inducing serious morphologic and functional deficits. While obvious tissue damage generally occurs after relatively high radiation doses, cognitive impairment can be seen after lower exposures. The mechanisms responsible for cognitive injury are not well understood, but may involve neurogenesis, a process that is affected by microenvironmental factors including oxidative stress and inflammation. In addition, damage to neurons, either directly or through environmental influences may have a profound impact on cognition. The relationships between cellular response, environmental factors and behavior are complex and difficult to study. However, understanding such issues should provide critical information relevant to the development of strategies and approaches to ameliorate or treat radiation-induced injuries that are associated with behavioral performance.

Accuracy of hippocampal network activity is disrupted by neuroinflammation: rescue by memantine.

Understanding how the hippocampus processes episodic memory information during neuropathological conditions is important for treatment and prevention applications. Previous data have shown that during chronic neuroinflammation the expression of the plasticity related behaviourally-induced immediate early gene Arc is altered within the CA3 and the dentate gyrus; both of these hippocampal regions show a pronounced increase in activated microglia. Low doses of memantine, a low to moderate affinity open channel uncompetitive N-Methyl-d-aspartate receptor antagonist, reduce neuroinflammation, return Arc expression to control levels and attenuate cognitive deficits induced by lipopolysaccharide. Here we investigate whether neuroinflammation affects the accuracy of information processing in the CA3 and CA1 hippocampal regions and if this is modified by memantine treatment. Using the immediate early gene-based brain-imaging method called cellular analysis of temporal activity by fluorescence in situ hybridization, it is possible to detect primary transcripts at the genomic alleles; this provides exceptional temporal and cellular resolution and facilitates the mapping of neuronal activity. Here, we use this method to compare the neuronal populations activated by two separate experiences in CA1 and CA3 and evaluate the accuracy of information processing during chronic neuroinflammation. Our results show that the CA3 pyramidal neuron activity is not stable between two exposures to the same environment context or two different contexts. CA1 networks, however, do not differ from control conditions. These data suggest that during chronic neuroinflammation, the CA3 networks show a disrupted ability to encode spatial information, and that CA1 neurons can work independently of CA3. Importantly, memantine treatment is able to partially normalize information processing in the hippocampus, suggesting that when given early during the development of the pathology memantine confers neuronal and cognitive protection while indirectly prevents pathological microglial activation.

Using superoxide dismutase/catalase mimetics to manipulate the redox environment of neural precursor cells.

Past work has shown that neural precursor cells are predisposed to redox sensitive changes, and that oxidative stress plays a critical role in the acute and persistent changes that occur within the irradiated CNS. Irradiation leads to a marked rise in reactive oxygen species (ROS) that correlates with oxidative endpoints in vivo and reductions in neurogenesis. To better understand the impact of oxidative stress on neural precursor cells, and to determine if radiation-induced oxidative damage and precursor cell loss after irradiation could be reduced, a series of antioxidant compounds (EUK-134, EUK-163, EUK-172, EUK-189) were tested, three of which possess both superoxide dismutase (SOD) and catalase activities and one (EUK-163) whose only significant activity is SOD. Our results show that these SOD/catalase mimetics apparently increase the oxidation of a ROS-sensitive fluorescent indicator dye, particularly after short (12 h) treatments, but that longer treatments (24 h) decrease oxidation attributable to radiation-induced ROS. Similarly, other studies found that cells incubated with CuZnSOD showed some increase in intracellular ROS levels. Subsequent data suggested that the dye-oxidising capabilities of the EUK compounds were linked to differences in their catalase activity and, most likely, their ability to catalyse peroxidative pathways. In unirradiated mice, the EUK-134 analogue induced some decrease of proliferating precursor cells and immature neurons 48 h after radiation, an effect that may be attributable to cytotoxicity and/or inhibition of precursor proliferation. In irradiated mice, a single injection of EUK-134 was not found to be an effective radioprotector at acute times (48 h). The present results support continued development of our in vitro model as a tool for predicting certain in vivo responses, and suggest that in some biological systems the capability to scavenge superoxide but produce excess H(2)O(2), as is known for CuZnSOD, may be potentially deleterious. Our results also show that the ability of catalase mimetics, like true catalases, to catalyse peroxidase reactions can complicate the interpretation of data obtained with certain fluorescent ROS-indicator dyes.

Apoptosis in the subependyma of young adult rats after single and fractionated doses of X-rays.

Ionizing radiation is commonly used in the treatment of brain tumors but can cause significant damage to surrounding normal brain. The pathogenesis of this damage is uncertain, and understanding the response of potential target cell populations may provide information useful for developing strategies to optimize therapeutic irradiation. In the mammalian forebrain, the subependyma is a mitotically active area that is a source of oligodendrocytes and astrocytes, and it has been hypothesized that depletion of cells from this region could play a role in radiation-induced white matter injury. Using a distinct morphological pattern of nuclear fragmentation and an immunohistochemical method to specifically label the 3'-hydroxyl termini of DNA strand breaks (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling), we quantified apoptosis in the subependyma in the young adult rat brain after single and fractionated doses of X-rays. Significant increases in apoptotic index (percentage of cells showing apoptosis) were detected 3 h after irradiation, and the peak apoptotic index was detected at 6 h. Six h after irradiation, the dose response for apoptosis was characterized by a steep increase in apoptotic index between 0.5 and 2.0 Gy and a plateau from 2-30 Gy. The fraction of cells susceptible to apoptosis was estimated to be about 40%, and treatment of rats with cycloheximide inhibited apoptosis. When daily 1.5-Gy fractions of X-rays were administered, the first three fractions were equally effective at decreasing the cell population via apoptosis. There was no additional apoptosis or decrease in cellularity in spite of one to four additional doses of X-rays. Those data suggested some input of cells into the subependymal population during fractionated treatment, and subsequent studies showed that there was a significant rise in 5-bromo-2' deoxyuridine labeling index 2-3 days after irradiation, indicating increased cellular proliferation. The proliferative response after depletion of cells via apoptosis may represent the recruitment of a relatively quiescent stem cell population. It is possible that the radiation response of subependymal stem cells and not the apoptotic-sensitive population per se are critical elements in the response of the brain to radiation injury.

Inhibition of dentate granule cell neurogenesis with brain irradiation does not prevent seizure-induced mossy fiber synaptic reorganization in the rat.

Aberrant reorganization of dentate granule cell axons, the mossy fibers, occurs in human temporal lobe epilepsy and rodent epilepsy models. Whether this plasticity results from the remodeling of preexisting mossy fibers or instead reflects an abnormality of developing dentate granule cells is unknown. Because these neurons continue to be generated in the adult rodent and their production increases after seizures, mossy fibers that arise from either developing or mature granule cells are potential substrates for this network plasticity. Therefore, to determine whether seizure-induced, mossy fiber synaptic reorganization arises from either developing or mature granule cell populations, we used low-dose, whole-brain x-irradiation to eliminate proliferating dentate granule cell progenitors in adult rats. A single dose of 5 Gy irradiation blocked cell proliferation and eliminated putative progenitor cells in the dentate subgranular proliferative zone. Irradiation 1 d before pilocarpine-induced status epilepticus significantly attenuated dentate granule cell neurogenesis after seizures. Two irradiations, 1 d before and 4 d after status epilepticus, essentially abolished dentate granule cell neurogenesis but failed to prevent mossy fiber reorganization in the dentate molecular layer. These results indicate that dentate granule cell neurogenesis in the mature hippocampal formation is vulnerable to the effects of low-dose ionizing irradiation. Furthermore, the development of aberrant mossy fiber remodeling in the absence of neurogenesis suggests that mature dentate granule cells contribute substantially to seizure-induced network reorganization.

Thermal distribution studies of helical coil microwave antennas for interstitial hyperthermia.

An implantable 915 MHz helical coil antenna was developed for improved localization and control of interstitial microwave hyperthermia. The radiating element consisted of a fine wire coil wound back over the inner conductor of a miniature semi-rigid coaxial cable in place of the terminal portion of outer conductor. The power deposition profiles from single helical coil antennas were studied both in homogeneous phantom and in muscle tissue in vivo and compared to those of single half-wavelength linear dipole antennas. The effects of variable coil length, turn density, and antenna insertion depth in tissue were characterized. The helical coil antennas produced a well-localized heating pattern with a sharp falloff of temperature in both directions axially from the coil element. One of the best heating patterns was obtained with a 35 turn, 35 mm long helical coil element which was separated from the antenna feedline outer conductor by a 1 mm gap (HCS-35(1)/36). This antenna showed a marked shift of the effectively heated volume toward the antenna tip and essentially no dependence of the heating pattern on insertion depth. In contrast, the axial power deposition profiles of dipole antennas were strongly affected by insertion depth and exhibited an inadequately heated area at the antenna tip even with 1/2-3/4 wavelength insertion. Thermal distribution studies showed that the single helical coil microwave antenna provided more predictable, well-localized heating of deep-seated tissues, with minimal requirement for over-implanting of the treatment volume.

Interstitial microwave hyperthermia in a canine brain model.

A dual frequency microwave system was constructed for interstitial heating of brain tissue. Single-junction dipole antennas were tested in a phantom model and in normal dog brain to determine how variations in physical factors affected temperature distributions. Non-survival studies were performed at both 915 and 2450 MHz to determine heating patterns that could be achieved within normal brain using this system. Chronic survival studies were performed using a single dipole antenna inserted laterally into one hemisphere of brain and driven at 2450 MHz. Temperatures of 43 or 44 degrees C for 30 min at a reference point 0.5 cm from the antenna junction were used to induce a thermal lesion of approximately 1 cm diameter in the right cerebral hemisphere of dogs. Neurologic and physical changes in dogs were monitored daily for up to 16 weeks after induction of cerebral lesions. The extent and development of thermal lesions was monitored with weekly computed tomographic (CT) examinations and, after death, at histopathologic examination. Results of the phantom studies showed that the longitudinal heating pattern was bell-shaped at both frequencies used and that there was some variation in heating length that depended on insertion depth. Acute studies in dog brain showed that 915 MHz antennas implanted less than 6.5 cm deep produced erratic heating patterns that usually included excessive heating of the surface of the brain. Conversely, 2 cm-long antennas driven at 2450 MHz gave reproducible temperature distributions both longitudinally along and radially away from the antenna. The steepest gradients--about 1 degree C/mm--occurred in the radial direction away from the antenna junction. A single 30 min heat treatment produced a large focal lesion that consisted of central coagulation necrosis surrounded by a sharply demarcated hypervascular zone. Edematous changes were minimal and were observed only during the first week after treatment. As assessed by serial CT scans, thermal lesions reached a maximum size by the first week after treatment and were essentially resolved by 16 weeks after treatment.

Microglial responses after focal radiation-induced injury are affected by alpha-difluoromethylornithine.

PURPOSE: The objective of this study was to quantify microglial and astrocytic cell responses after focal 125I irradiation of normal brain and to determine the effects of an intravenous infusion of alpha-difluoromethylornithine (DFMO) on those responses. METHODS AND MATERIALS: Adult beagle dogs were irradiated using high activity 125I sources. Saline or DFMO (75 mg/kg/day) was infused for 18 days, and 1 to 10 weeks later brain tissues were collected. Immunohistochemical stains were used to label phagocytes and amoeboid microglia (lectin RCA-1), astrocytes (GFAP), and cells synthesizing deoxyribonucleic acid (DNA) (BrdU). Cell densities (cells/mm2) and BrdU labeling indices were quantified. RESULTS: In dogs infused with saline, increases in phagocytes and amoeboid microglia were observed at 1-2 weeks and 4 weeks, respectively. The labeling indices for phagocytes and amoeboid microglia peaked at 4 weeks with maximum values of 4.8 and 13.4%, respectively. Astrocyte cell numbers increased from 2-6 weeks following irradiation; increased labeling indices were observed after 2 weeks. An infusion of DFMO significantly suppressed BrdU labeling and delayed the increase in cell numbers for phagocytes and amoeboid microglia. In both treatment groups, the proportion of total BrdU labeling accounted for by phagocytes was maximum 1 week after irradiation and then decreased. The proportion of total BrdU labeling accounted for by amoeboid microglia and astrocytes was zero for 2 weeks and then increased. CONCLUSIONS: Microglial reactions after focal irradiation involve the phagocytic and amoeboid cell forms and are characterized by increased BrdU uptake and increased cell number. DFMO significantly alters these responses. Changes in astrocyte cell number and BrdU labeling may be related to changes in microglia. Studies of cell responses and their modification may lead to a better understanding of the pathogenesis of radiation injury, and to new strategies to optimize the use of therapeutic irradiation.

Radiation dose response of normal brain.

Dose response relationships were determined after hemibrain x-irradiation of normal beagle dogs. Radiation doses of 11.5, 13.5, 14.3, and 17 Gy were delivered in a single dose and results were compared to previous studies using doses of 15 and 30 Gy. Brain injury was quantified using computed tomography (CT), with serial studies obtained monthly up to 1 year following irradiation. Quantitative endpoints included low density volume and contrast enhancement. Doses above 14.3 Gy resulted in high lethality 5-8 months following irradiation, and an LD50 of 14.9 Gy was calculated. At these lethal doses, low density volume representing edema, demyelination, and necrosis had a similar response with an ED50 of 14.6 Gy. Radiation-induced decreases in white matter density appeared 5-6 months after sublethal doses (less than or equal to 14.3 Gy) and the volume of tissue characterized by this low density increased with time and dose. This sublethal low density change had an ED50 of 12.8 Gy, and may reflect a loss or generalized atrophy of glial cells and/or myelin. These results show that: (a) the dose response curves obtained after hemibrain x-irradiation are extremely steep; and (b) at least two processes may be involved in the development of late radiation damage, one that is rapid upon onset (a "delayed acute" reaction) and the other which is a slower and more degenerative process.

Cellular proliferation and infiltration following interstitial irradiation of normal dog brain is altered by an inhibitor of polyamine synthesis.

PURPOSE: The objectives of this study were to quantitatively define proliferative and infiltrative cell responses after focal 125I irradiation of normal brain, and to determine the effects of an intravenous infusion of alpha-difluoromethylornithine (DFMO) on those responses. METHODS AND MATERIALS: Adult beagle dogs were irradiated using high activity 125I sources. Saline (control) or DFMO (150 mg/kg/day) was infused for 18 days starting 2 days before irradiation. At varying times up to 8 weeks after irradiation, brain tissues were collected and the cell responses in and around the focal lesion were quantified. Immunohistochemical stains were used to label astrocytes (GFAP), vascular endothelial cells (Factor VIII), polymorphonuclear leukocytes (PMNs; MAC 387) and cells synthesizing deoxyribonucleic acid (DNA) (BrdU). Cellular responses were quantified using a histomorphometric analysis. RESULTS: After radiation alone, cellular events included a substantial acute inflammatory response followed by increased BrdU labeling and progressive increases in numbers of capillaries and astrocytes. alpha-Difluoromethylornithine treatment significantly affected the measured cell responses. As in controls, an early inflammatory response was measured, but after 2 weeks there were more PMNs/unit area than in controls. The onset of measurable BrdU labeling was delayed in DFMO-treated animals, and the magnitude of labeling was significantly reduced. Increases in astrocyte and vessel numbers/mm2 were observed after a 2-week delay. At the site of implant, astrocytes from DFMO-treated dogs were significantly smaller than those from controls. CONCLUSIONS: There is substantial cell proliferation and infiltration in response to interstitial irradiation of normal brain, and these responses are significantly altered by DFMO treatment. Although the precise mechanisms by which DFMO exerts its effects in this model are not known, the results from this study suggest that modification of radiation injury may be possible by manipulating the response of normal cells to injury.

X-irradiation causes a prolonged reduction in cell proliferation in the dentate gyrus of adult rats.

The effects of X-irradiation on proliferating cells in the dentate subgranular zone were assessed in young adult Fisher 344 rats exposed to a range of X-ray doses and followed for up to 120 days. Apoptosis was quantified using morphology and end-labeling immunohistochemistry, and cell proliferation was detected using antibodies against the thymidine analog BrdU and the cyclin-dependent kinase p34(cdc2). Radiation-induced apoptosis occurred rapidly, with maximum morphological and end-labeling changes observed 3-6h after irradiation. Twenty-four hours after irradiation cell proliferation was significantly reduced relative to sham-irradiated controls. The number of apoptotic nuclei increased rapidly with radiation dose, reaching a plateau at about 3Gy. The maximum number of apoptotic nuclei was substantially higher than the number of proliferating cells, suggesting that non-proliferating as well as proliferating cells in the subgranular zone were sensitive to irradiation. Subgranular zone cell proliferation was significantly reduced relative to age-matched controls 120 days after doses of 5Gy or higher. These findings suggest that neural precursor cells of the dentate gyrus are very sensitive to irradiation and are not capable of repopulating the subgranular zone at least up to 120 days after irradiation. This may help explain, in part, how ionizing irradiation induces cognitive impairments in animals and humans.

Relative uptake of low- and high-osmolality contrast media in CT of brain tumors.

The magnitude and time course of contrast enhancement in spontaneous canine brain tumors was determined for two contrast agents: meglumine iothalamate and sodium meglumine ioxaglate. Tumor enhancement during contrast infusion and at 5, 10, 15, 30 and 45 minutes was measured using quantitative computed tomography. Blood iodine was measured using x-ray fluorescence. Peak contrast enhancement occurred during the infusion, and the magnitude was the same for both agents. Per gram of iodine infused, blood iodine was 12.4% higher with ioxaglate than iothalamate. The monoionic dimer ioxaglate is as effective as iothalamate for enhancement of canine brain tumors.

The radioresponse of the central nervous system: a dynamic process.

Radiation continues to be a major treatment modality for tumors located within and close to the central nervous system (CNS). Consequently, alleviating or protecting against radiation-induced CNS injury would be of benefit in cancer treatment. However, the rational development of such interventional strategies will depend on a more complete understand-ing of the mechanisms responsible for the development of this form of normal tissue injury. Whereas the vasculature and the oligodendrocyte lineage have traditionally been considered the primary radiation targets in the CNS, in this review we suggest that other phenotypes as well as critical cellular interactions may also be involved in determining the radio-response of the CNS. Furthermore, based on the assumption that the CNS has a limited repertoire of responses to injury, the reaction of the CNS to other types of insults is used as a framework for modeling the pathogenesis of radiation-induced damage. Evidence is then provided suggesting that, in addition to acute cell death, radiation induces an intrinsic recovery/repair response in the form of specific cytokines and may

Cerebrovascular response after interstitial irradiation.

To characterize the role of the cerebrovascular response in the development of brain injury after focal irradiation, 125I sources were implanted in frontal white matter of the brain of normal dogs; dose was 20 Gy, 7.5 mm from the source. Cerebral blood flow, vascular volume and mean transit time of blood were quantified in irradiated tissues relative to tissues in the contralateral hemisphere and analyzed with respect to previously determined volumetric measurements of damage and the blood-to-brain transfer constant. Blood flow and vascular volume within the radiation-induced focal lesion were maximally reduced 3 weeks after implant, when necrosis volume was maximal. By 6 weeks, vascular volume and mean transit time were increased, suggesting a strong neovascular response. In tissues surrounding the lesion, blood flow and vascular volume were reduced 1-4 weeks after irradiation and approached normal at 6 weeks; average mean transit time was not altered significantly. Alterations in blood flow and mean transit time were significantly related to edema volume and transfer constant, but alterations in vascular volume were not, suggesting that edema-induced vascular compression was not responsible for changes in blood flow. Reductions of radiation-induced permeability of the blood-brain barrier and/or edema might limit radiation-induced changes in blood flow and the extent of tissue injury.

Modification of radiation-induced brain injury by alpha-difluoromethylornithine.

The effect of alpha-difluoromethylornithine (DFMO) on 125I-induced brain injury was investigated in a dog model. Cerebrospinal putrescine levels were reduced from baseline levels 1-2 weeks after irradiation in animals treated with 125I and DFMO, while putrescine levels were elevated in 125I and saline-treated animals. In addition, the time course of changes in the volumes of edema, necrosis, and tissue showing evidence of blood-brain barrier breakdown was altered significantly by DFMO treatment. The most significant alterations occurred 2-4 weeks after irradiation, at which times the average volumes of damage in DFMO-treated animals were reduced compared to saline-treated animals. The time course of alterations in blood-to-brain transfer, brain-to-blood transfer, and vascularity following irradiation was also altered by DFMO treatment. Analysis of variance demonstrated a strong relationship of blood-to-brain transfer and vascularity to volume of edema, suggesting that the effect of DFMO on edema may be partially mediated by its effects on blood-brain barrier breakdown.

Pathology of delayed radiation brain damage: an experimental canine model.

Delayed radiation damage of normal brain can be a devastating complication of radiation therapy and generally occurs months to years after the initiation of therapy. Primarily restricted to the white matter, radiation damage is characterized by a number of histopathologic changes including coagulation necrosis, vascular alterations with fibrinoid necrosis, edema, and demyelination. Normal dogs were exposed to either 10, 15, or 30 Gy of X rays to a single hemisphere and the gross and histopathologic changes were evaluated qualitatively. A spectrum of changes was observed ranging from white matter edema to extensive white matter necrosis, and the extent, location, and type of damage were dependent upon radiation dose. Histopathologic changes were separated into three major categories based on the character and size of the lesions, with the most severe changes being similar to the types of changes described in human patients who have developed delayed radiation necrosis. Less severe forms of damage such as multifocal, sometimes confluent areas of microscopic necrosis with spongiotic borders and edema with severe axonal swelling were also observed. These latter changes are not well recognized as being due to radiation. The findings of this study also indicate that many of the changes ascribed to combined treatment with methotrexate and radiation in humans are induced in the normal dog brain by radiation alone. The results of his study show that the dog is a suitable model of the human brain for studying radiation brain injury and may be useful for investigation of drug-radiation interactions.

Radiation brain injury is reduced by the polyamine inhibitor alpha-difluoromethylornithine.

Alpha-difluoromethylornithine (DFMO) was used to reduce 125I-induced brain injury in normal beagle dogs. Different DFMO doses and administration schedules were used to determine if the reduction in brain injury was dependent on dose and/or dependent upon when the drug was administered relative to the radiation treatment. Doses of DFMO of 75 mg/kg/day and 37.5 mg/kg/day given 2 days before, during and for 14 days after irradiation reduced levels of putrescine (PU) in the cerebrospinal fluid relative to controls. Volume of edema was significantly reduced by 75 mg/kg/day of DFMO before, during and after irradiation and by the same dose when the drug was started immediately after irradiation. A reduction in edema volume after 37.5 mg/kg/day before, during and after irradiation was very near significance. Ultrafast CT studies performed on dogs that received a DFMO dose of 75 mg/kg/day before, during and after irradiation suggested that the reduced edema volume was associated with reduced vascular permeability. Volume of necrosis and volume of contrast enhancement (breakdown of the blood-brain barrier) were significantly lower than controls only after a DFMO dose of 75 mg/kg/day before, during and after irradiation. These latter data, coupled with the findings relative to edema, suggest that different mechanisms may be involved with respect to the effects of DFMO on brain injury, or that the extents of edema, necrosis and breakdown of the blood-brain barrier may depend upon different levels of polyamine depletion. The precise mechanisms by which DFMO exerts the effects observed here need to be determined.

Computed tomography analysis of the canine brain: effects of hemibrain X irradiation.

Radiation damage induced by megavoltage X irradiation in normal brain tissue can manifest as one of several pathologic processes depending upon the time of brain examination after irradiation. Serial quantitative computed tomography (QCT) analyses were used to study the development of radiation damage in the normal canine brain. Tissue density, volume of low density areas, magnitude and volume of contrast uptake, and ventricular volume were measured following hemibrain irradiation and were correlated with histopathology. Low density areas correlated with edema, demyelination, axonal swelling, and necrosis and appeared 3-4 months after irradiation. Large regions of contrast enhancement (coagulation necrosis and associated vascular changes) appeared 5-6 months after irradiation. Results from this study demonstrated that the pathologic changes induced in the dog brain after single doses of X rays were similar to the changes observed in nonhuman primates and man after exposure to radiation.

Measurement of vascular permeability in spinal cord using Evans Blue spectrophotometry and correction for turbidity.

Vascular permeability can be visualized by Evans Blue (EB) extravasation and quantified by spectrophotometry after formamide extraction of the tissue. However, formamide extracts show significant turbidity, which may contribute to the total optical density at the wavelength of measurement (e.g., 620 lambda). We developed a simple method for estimating the component of the total optical density of a dyed specimen contributed by turbidity. Our method, which uses a determination of turbidity made at another point of the light spectrum (740 lambda), was more precise than two other EB quantification techniques. We therefore recommend it for individual correction of formamide extracts of spinal cord specimens. The application of this technique to the brain remains to be determined.

Apoptosis is induced in the subependyma of young adult rats by ionizing irradiation.

To determine if radiation-induced apoptosis occurred in young adult brain, we exposed 2-3-month old rats to single x-ray doses of 5 or 30 Gy. Apoptosis was quantified using the TdT-mediated dUTP-biotin nick end labeling (TUNEL) method and a morphologic assessment of nuclear fragmentation. Apoptosis occurred primarily in the subependyma but also in the corpus callosum, peaking 6 h after irradiation. At 48 h there were no apoptotic nuclei observed. These data are the first to show that apoptosis occurs in the young adult rat brain after ionizing irradiation. Further studies are required to define the particular cell type(s) involved and to address the role of this process in the pathogenesis of late radiation injury.