Neuronal Injury After Photoactivation of Photofrin 11 - Europe PMC

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Radiation Oncology,t and Neurology,* Henry Ford Hospital,. Detroit, Michigan, and Oakland University,l. Rochester, Michigan. Photodynamic therapy has been ...
American Journal of Pathology, Vol. 141, No. 4, October 1992 Copyright X) American Association of Pathologists

Neuronal Injury After Photoactivation of Photofrin 11 Yasuji Yoshida,* Mary 0. Dereski,t Julio H. Garcia,* Frederic W. Hetzel,t and Michael Chopptt" From the Departments of Pathology and Neuropathology,* Radiation Oncology,t and Neurology,* Henry Ford Hospital, Detroit, Michigan, and Oakland University,l Rochester, Michigan

Photodynamic therapy has been used in the management ofpatients with malignant brain tumors even though the effects of this form of treatment on the adjacent normal brain are incompletely characterized. The authors examined, in sequential experiments, morphologic alterations affecting the cerebral cortex in rats injected with Photophrin II and exposed to light Initially, minimal cell alterations, including cisternal swelling of both endoplasmic reticulum and Golgi apparatus, involved only neurons located in the superficial layers of the cerebral cortex exposed to light These changes spread, over a period of several hours, from the surface to the bottom of the cortex and eventually involved the entire cortical segment exposed to light The earliest structural signs of lethal injury to neurons developed over a period of 18 hours after porphyrns had been photoactivated and astrocytes had been severely damaged. Signs of lethal injury to neurons included an increase in the number of mitochondrial cristae and appearance of amorphous electron-dense deposits within swollen mitochondria. The appearance of these alterations was followed by segregation of intracytoplasmic organelles and fragmentation of nuclear and cytoplasmic membranes. The tissue changes, including those involving neurons, eventually progressed to coagulation necrosis at 48 hours. These observations suggest that prophyrins injected to rats (48 hours before photoactivation) cause swelling and necrosis of astrocytes. This is followed by neuronal necrosis, which appears at two time intervals; the initial neuronal necrosis occurs after the astrocytic disintegration A second type of neuronal alteration appears after microvessels become thrombosed and ischemia is likely to develop. (Am J Pathol 1992, 141:989-99 7)

Acute neuronal injury in models of focal ischemia has been widely studied by physiologic means1' and less extensively by ultrastructural methods.' Irreversible neuronal injury in areas of focal ischemia may develop over several hours, as suggested by reperfusion experiments in subhuman primates with middle cerebral artery (MCA) occlusion.910 The events leading to the necrosis of neurons injured by ischemia are multiple, complex, and incompletely characterized4'11; several lines of evidence suggest that free radical formation may be an important contributor to the induction of neuronal necrosis in ischemia, especially during reperfusion.11-14 Photosensitizing agents, such as Photofrin 11, cause necrosis of cells (neoplastic as well as normal), probably by promoting formation of free radical species, on photoactivation.15.16 We completed a time-dependent study (1 hour to 7 days) of the effects that photoactivating Photofrin 11 may have on normal rat brain and reported early injury to astrocytes and, secondarily, to the microvasculature and the neurons.17 In the current analysis, we examine closely the sequential nature of the neuronal alterations. The objective is to compare the structural alterations of neuronal perikarya at the site of brain injury, induced by photoactivated Photophrin 11,17.18 with brain lesion secondary to a single artery occlusion.10 This comparison may provide useful clues on mechanisms responsible for inducing neuronal necrosis at a site of focal ischemia. The information derived from these experiments also could influence the design of strategies that eventually may lead to wider application of this form of therapy (PDT) to the management of patients with brain tumors.19

Materials and Methods Forty-eight adult male Fisher rats (200 to 250 g) were used. One experimental group (n = 38) and two control groups (n = 10) were prepared. All rats in the three Supported by NIH grant P01-CA 43892. Accepted for publication Aprl 20,1992. Address reprint requests to Dr. J. H. Garcia, Department of Pathology, K-6, Henry Ford Hospital, 2799 W. Grand Boulevard, Detroit, Ml 48202-2689.

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Figure 1. Rat brain, 24 hours after PDT; the lesion is well demarcated, limited to the site ofphotoactivation and surrounded by a peripheral zone ofpallor (x 4).

except for four control rats that were treated with laser alone, were injected intraperitoneally with 12.5 mg/ kg sterile Photofrin 11 (Quadralogic Technologies Ltd, Vancouver, Canada), pH 7.23, osmolarity 179 mOsm/kg. groups,

Forty-eight hours later, all rats were anesthetized intraperitoneally with ketamine (44 to 80 mg/kg) and zylaxine (13 mg/kg). Atropine (0.04 mg/kg) also was injected at the time of anesthesia. The methods for craniectomy and details of the laser treatment have been described elsewhere.2' Briefly, the rat's scalp was shaved and a midline incision was made. A 5.0-mm diameter craniectomy exposed the dura from the midline over the right hemisphere at a site located next to the coronal suture; the laser beam was shone over the intact dura mater over a period of 5 minutes 22 seconds. Core temperature in all rats was maintained (370C) with a recirculating pad and K module and monitored through intrarectal type T module. An Argon-pumped dye laser (Coherent Radiation, Palo Alto, CA) was used at a wavelength of 632 ± 2 nm. The fiberoptic was placed approximately 10 mm from the dural surface, and energy was maintained at 100 mW/cm2. Brain temperature was monitored during exposure to the laser with a thermocouple inserted in the brain at the pe.A; -¢ ^v.

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Figure 2. All sections are from the cerebral cortex, 1.0 p.m thick, and stained with toluidine blue; x460. A: Cerebral cortex from one of the control animals. B: One hour after PDT; marked astrocytic swelling and intact neurons. C: Eighteen hours after PDT most neurons show hyalinized cytoplasm and pyknosis; there is marked neuropil sponginess. D: Seven days after PDT coagulation necrosis is apparent; there are many ghost cells, and

the neuropil is disorganized.

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riphery of the craniectomy site. All animals were treated with 35 J/cm2 of optical energy. After exposing the dura to laser, the scalp was sutured closed and animals were observed for several hours after surgery. Four to seven rats were killed at each of the eight time intervals (1, 4, 6, 12, 18, 24, 48 hours, and 7 days after the laser treatment). Under general anesthesia, each rat was transcardially perfused with either 4% paraformaldehyde (PA) in 0.1 mol/l (molar) phosphate buffer (PB) for routine histologic and immunohistochemical studies, or with 3% glutaraldehyde (GA) in 0.1 mol/l PB for electron microscopy. The brains fixed in 4% PA were removed and immersed overnight in the same fixative kept at 4°C. After

fixation, brains were coronally cut into slices of 2.0-mm thickness. All slices, including the area irradiated with the laser, were routinely processed and embedded in paraffin. One of the coronal slices containing the lesion was immersed in 3% GA in 0.1 mol/l PB and prepared for electron microscopic examination. Brains fixed in 3% GA in 0.1 mol/l PB were cut into slices 2.0 mm thick, and trimmed into sample pieces (1.0 x 1.0 x 2.0 mm). These samples were posifixed in 1% osmium tetroxide for 3 hours, dehydrated through graded ethanols, and sections 1.0 ,. thick were stained with toluidine blue. Ultrathin sections of the area of interest were prepared, doublestained with uranyl acetate and lead citrate following the

Figure 3. A: A neuron in the cerebral cortex 6 hours after PDT, shows minimal changes (cisternal dilatations); in contrast, there is definite swelling ofperineuronal astrocytic processes, x 4,600. B: At 12 hours after PDT, the neuronal cytoplasm is electron lucent and contains dispersed cell organelles; however, mitochondria and cell membranes are preserved, x 6,600.

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Reynold's method, and examined with a Philips 300 electron microscope (80 kV). Two groups of animals were used as controls; rats treated with Photofrin 11 but not exposed to laser were killed at 1 and 24 hours after surgery; rats in the second control group, exposed to laser but not to Photofrin 11, were killed one each at 1, 24, 48 hours, and 7 days after the surgery. In the experimental group, the contralateral cerebral hemisphere also served as a source of control tissues that were exposed to Photofrin II but not to laser. The reported results are based on a meticulous analysis of about 750 histology slides and 700 electron micrographs.

perikaryal neurofilaments and microtubules, and discontinuous plasma membranes were easily recognized (Figures 4, 5A, B). Mitochondrial configuration was preserved, although increasing numbers of amorphous matrical densities appeared while most RER cisternae adopted a small, round configuration (Figure 5). At 48 hours to 7 days after PDT, the area at risk was markedly pale (compared with the surrounding tissues), neuropil became vacuolated, and many eosinophilic neurons showed early nuclear disintegration (Figures 6A, 6B, 7) Some neurons retained their pyramidal shape; the contour of swollen astrocytes, blood vessels, and neurons was recognizable despite the loss of affinity for he-

Results Control animals treated with either Photofrin 11 alone or exposed to laser only showed no histologic or ultrastructural abnormalities, except for a minute lesion that resulted from the craniectomy and the insertion of the thermocouple used to monitor the brain temperature. In all experimental animals, the lesions described below were confined to the cortex exposed to the laser (Figure 1). One hour after PDT, there were some perivascular and perineuronal vacuoles in the neuropil of the superficial cortical layers (layers and 111); these vacuoles correspond to swollen astrocytic perikarya and astrocytic processes; the intrinsic neuronal features were intact (Figure 2B). Four hours after PDT, cortical neurons often showed "scalloping" as a consequence of the swelling of numerous perineuronal astrocytic processes. Intrinsic neuronal alterations were still minimal, however, and included only mild dilatation of the rough endoplasmic reticulum (RER) and the Golgi apparatus cisternae; neuronal mitochondria were unremarkable. A few presynaptic terminals in the superficial layers of the cortex also showed watery swelling. Neurons located in the deep layers of the cerebral cortex were entirely unremarkable. At 6 to 12 hours, the affected neurons in the superficial cortical layers showed cisternal dilatation, decreasing number of polysomes, and segregation of organelles in the perikaryon. Nuclear chromatin and nucleolus in these neurons were segregated; however, neuronal mitochondria showed intact matrix and cristae, whereas astrocytic mitochondria appeared condensed (Figure 3A, B). Neuronal changes of this type were less prominent in the deep cortical layers. At 18 to 24 hours after PDT, the irradiated area was edematous, pale, and clearly demarcated from the surrounding brain tissue (Figure 1). Most neurons were eosinophilic and contained pyknotic nuclei and vacuolated nuclear membranes; segregated cell organelles, loss of

..-;*,-Y,' Figure 4. At 18 hours after PDT, a neuron in the lesion shows absence of neurofilaments and microtubules; there is disorganization of markedly dilated organelles. Deposits of electron-dense materials in swollen mitochondria with increased matrical density indicate lethal damage to the cell, x 6,000.

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Figure 5. A. At 24 hours, a neuron from the site ofphotoactivation shows nuclear alterations and diruption of cytoplasmic organelles, x 7,500 B: A degenerating cell, probably a swollen astrocyte, also shows chromatin clumping and disorganization of cytoplasmic organelles, X8,700.

matoxylin. (Figure 2D). In these cells, the mitochondrial shape was preserved although many displayed increased number of cristae, and electron-dense matrical densities (Figure 6A, B). At the margins of the cortical area exposed to light, there was infiltration by macrophages; neutrophils, reactive astrocytes, and proliferated blood vessels also became noticeable. A different type of neuronal change appeared in this marginal area; it consisted of cytoplasmic condensation and marked mitochondrial swelling (Figure 6C, D). In summary, the features of neuronal injury induced by PDT include: 1) the changes spread, over a period of hours, from the surface to the bottom of the cortex, and

also in a centrifugal manner; 2) the structural signs of neuronal necrosis appear first 18 hours after the photoactivation of porphyrins; 3) this delayed change consists of an increased number of neuronal mitochondria cristae and appearance of amorphous matrical densities in the swollen mitochondria; at later times, we noted segregation of intracytoplasmic organelles and disruption of the nuclear and plasma membranes; 4) the neuronal changes spread uniformly throughout the cortex exposed to the laser, but this alteration was preceded by astrocytic and endothelial degeneration; changes involving all cell types, within the area at risk, progressed to coagulation necrosis 48 hours after PDT.

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Figure 6. At 48 hours after PDT, two kinds of neuronal changes are observed. A: A neuron in the center of the lesion shows a 'ghost" appearance in which nuclear and cytoplasmic contours are still discernible. At higher magnification (B), injured mitochondria show prominent electron densities and increased electron density matrix. A, X9,200, B, X26,000; C: A cortical neuron located at the margin of the area at risk shows acute degenerative changes with cisternal dilatation of RER and Golgi apparatus. There are also swollen, exploded mitochondria, without electron-dense deposits (D). C, x6,300; D, 17,000.

Discussion This study showed characteristic changes in neurons located at the cortical site directly exposed to light. There was no significant change during the initial 12 hours; during the period between 18 to 48 hours, cisternal dilatation and neuronal mitochondrial alterations became increasingly worse. The mitochondrial changes included matrical deposition of amorphous densities that were structurally identical to those identified in several studies of acute cell injury as being calcium carbonate deposits.2'22 Such electron-dense deposits were most easily found 7 days after PDT, when the lesion reached its acme. Swollen, nearly exploded mitochondria have been described (as an early cellular change) in lymphoma cells exposed in vitro to photoactivated porphyrins23 and in neurons and astrocytes, during the early stages of focal ischemic brain injury.24 This type of change was visible in the PDT preparations, only as a delayed effect that was first noted 48 to 72 hours after the porphyrin activation. This delayed type of neuronal injury was confined to neurons located in the marginal area surrounding the cortex exposed to the laser, where ischemia secondary to microvascular thrombosis is likely to have developed. Neuronal changes at the site of PDT lesions seemingly proceed through two phases: the initial one (18 hours) is attributed to the cytotoxic effects of PDT; these effects were probably responsible for the swelling and

necrosis of astrocytes that occurred before neuronal necrosis was apparent.17 In addition to the mitochondrial deposits of electron-dense material, there were vacuolar changes affecting RER and Golgi cisterns, disappearance of neurofilaments and microtubules as well as disruption of nuclear and plasma membranes. These cellular changes, which are expressions of irreversible injury,25 began involving isolated neurons only 18 hours after PDT and proceeded to coagulation necrosis about 30 hours later. The cytotoxic effects of PDT have been attributed to metabolic cell reactions initiated by photoactivated porphyrins.26 The timing of the observations made in this study involving first astrocytes and then neurons, as well as previously published studies of altered brain metabolism using nuclear magnetic resonance spectroscopy27 are in keeping with that hypothesis. The time (4 hours) when changes in adenosine triphosphate metabolites develop and local CBF values are lowered28 coincides with the time when increasing numbers of swollen astrocytes and large numbers of thrombosed intraparenchymal microvessels become detectable.17 The secondary or delayed effect (confined to the periphery of the area at risk) probably has an ischemic cause. Dietrich et a128 described focal brain necrosis, induced by photoactivation of rose bengal; in this model of brain necrosis, intravascular thrombosis occurs within a few minutes and quickly becomes widespread. Local

IL WI ;'^) " Figure 7. Seven days after PDT a ghost cell (probably a neuron) is surrounded by cytoplasmicfragments of inflammatory cells, X8,500.

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cerebral blood flow values drop, especially at the site of photoactivation, and the ischemic effects persist for 5 days after the induction of the focal brain lesion.28 Neuronal changes at sites of focal brain ischemia are said to be closely related to the increased concentration of active oxygen species (oxygen radicals).11 These radicals are unstable and extremely reactive with other compounds, especially polyunsaturated fatty acids of lipid bilayers both in cytoplasmic organelles and in plasma membrane; as a result, free radicals can mediate the disintegration of mitochondria and plasma membranes.14'25 We compared the lesions induced by PDT in rats with the structural abnormalities noted in models of focal brain ischemia. Eosinophilic neurons, progressive swelling of neuronal mitochondria, and irregular enlargement of the nuclear membranes were first noted at 3 hours in rhesus monkeys with MCA occlusion6; these changes became more pronounced with time, although quantitation of the histologic changes was not attempted. Scattered eosinophilic neurons were initially detected in the brains of cats and monkeys, 7 hours after MCA occlusion.' Other changes indicative of lethal injury had begun after 15 minutes, in a few scattered cells, and these changes were thought to worsen with time; however, sequential time-dependent studies of histologic alterations developing after occlusion of a single artery have not been published. In experiments based on the transient occlusion (1 to 2 hours' duration) of a MCA followed by 24 hours' reperfusion, neuronal eosinophilia and other signs of neuronal necrosis were first recognized 18 hours after the MCA occlusion.10 Some similarities exist between timing of lethal injury in focal ischemia and the delayed (18 hours) neuronal injury of PDT. But, in sharp contrast with the nature of the injury secondary to activation of porphyrins, the most prominent feature of the histologic changes in regional brain ischemia is the heterogeneous and multifocal nature of the initial structural changes; this applies not only to the topographic distribution of the lesions but also to the time-dependent changes affecting various cell types.' Some of the neuronal changes in focal ischemia may be secondary to alterations affecting membranous phospholipids as a result of lipid peroxidation and degradation induced by oxygen-derived free radicals.' 1,12,25 OXygen-derived free radicals generated by the direct infusion of xanthine oxidase/hypoxanthine/ADP-Fe3+ system into the rat caudate-putamen induces, within 2 hours after the injection, spongy neuropil, and neuronal cytoplasmic vacuoles.30 Ultrastructural changes in the molecular layer of these brain slices included swelling of cellular processes and mitochondrial changes31 that, although unspecific, are seen both in focal ischemia and PDT injury. Forebrain ischemia in rats, secondary to transient (5.0 minutes) bilateral common carotid artery clamping, in-

duces either ischemic or necrotic changes in neurons. The appearance of one or the other is determined by topographic factors and by the length of time elapsed after the ischemic event.32 An unidentified factor (possibly glutamate), may be responsible for the hippocampal necrosis that develops in this model, but that occurs only during the period of reperfusion.Y Brown and Brierley3 described a different kind of neuronal change in rat hippocampus; this neuronal alteration (microvacuolation) resulted from an injury secondary to unilateral carotid ligation and asphyxia (exposure to nitrogen atmosphere); the neuronal changes these authors described in the hippocampus of these animals reflect the combined effects of ischemia, hypoxemia, and seizures. The biologic effects of seizures in rat brains are significantly different from those of focal ischemia in terms of the underlying neurochemical changes, the neuronal revival time, the time course of neuronal death, the distribution of selective neuronal necrosis, and the type of excitotoxins released.34 The reperfusion of the forebrain ischemia model, with its delayed neuronal necrosis, and the hypoxemia/seizures of the model used by Brown and Brierley, are elements missing from the conditions created by occluding a single artery. For these reasons, comparisons among these various conditions must be cautiously conducted. Neuronal damage in PDT lesions could be mediated by the neuronal incorporation of Photofrin 11, by breakdown of mechanisms dependent on astrocytic integrity, by ischemic changes resulting from diffuse microvascular thrombosis, or by a combination of all these factors. The time course and the nature of the mitochondrial changes recorded 18 hours after photoactivation suggest that the initial neuronal changes are mediated by astrocytic injury. Peng et a135 reported colocalization of Photofrin 11 and aluminum phthalocyanine tetrasulfonate in mouse astroglia, and we noted that after photoactivation astrocytes progressively degenerate whereas neurons remain relatively intact for the first 18 hours.17 The ultimate brain lesion induced by PDT and by focal ischemia appears identical: coagulation necrosis; this is not surprising, as it known that this is not a specific form of tissue response.25 In brain infarcts induced by a singleartery occlusion, however, the initial structural changes involve simultaneously astrocytes (swelling) and neurons (shrinkage and swelling).6'29 Moreover, the early changes of focal brain ischemia are not characterized by widespread microvascular thrombosis, and this is a very consistent feature of the PDT injury.17'36

Acknowledgments The authors thank Ms. Lisa Pietrantoni, HTL (ASCP), for assisting with histologic preparations, Ms. Barbara Caracciolo for prepar-

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ing the manuscript, and Quadralogic Technologies Ltd. for supplying the drug.

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