Time-dependent surface adhesive force and morphology of RBC measured by AFM

Atomic force microscopy (AFM) is a rapidly developing tool recently introduced into the evaluation of the age of bloodstains, potentially providing legal medical experts useful information for forensic investigation. In this study, the time-dependent, morphological changes of red blood cells (RBC) under three different conditions (including controlled, room-temperature condition, uncontrolled, outdoor-environmental condition, and controlled, low-temperature condition) were observed by AFM, as well as the cellular viscoelasticity via force-vs-distance curve measurements. Firstly, the data indicate that substrate types have different effects on cellular morphology of RBC. RBC presented the typical biconcave shape on mica, whereas either the biconcave shape or flattened shape was evident on glass. The mean volume of RBCs on mica was significantly larger than that of cells on glass. Surprisingly, the adhesive property of RBC membrane surfaces was substrate type-independent (the adhesive forces were statistically similar on glass and mica). With time lapse, the changes in cell volume and adhesive force of RBC under the controlled room-temperature condition were similar to those under the uncontrolled outdoor-environmental condition. Under the controlled low-temperature condition, however, the changes in cell volume occurred mainly due to the collapse of RBCs, and the curves of adhesive force showed the dramatic alternations in viscoelasticity of RBC. Taken together, the AFM detections on the time-dependent, substrate type-dependent, environment (temperature/humidity)-dependent changes in morphology and surface viscoelasticity of RBC imply a potential application of AFM in forensic medicine or investigations, e.g., estimating age of bloodstain or death time.     

Enhancement of ultrasonically induced cell damage by phthalocyanines in vitro

In this work, erythrocytes from carp were used as a nucleated cell model to test the hypothesis that the phthalocyanines (zinc - ZnPc and chloroaluminium -AlClPc) enhance ultrasonically induced damage in vitro. In order to confirm and complete our earlier investigation, the influence of ultrasound (US) and phthalocyanines (Pcs) on unresearched cellular components, was studied. Red blood cells were exposed to 1 MHz continuous ultrasound wave (0.61 and/or 2.44 W/cm²) in the presence or absence of phthalocyanines (3 μM). To identify target cell damage, we studied hemolysis, membrane fluidity and morphology of erythrocytes. To demonstrate the changes in the fluidity of plasma membrane we used the spectrofluorimetric methods using two fluorescence probes: 1-[4-(trimethylamino)phenyl]-6-phenyl-1,3,5,-hexatriene (TMA-DPH) and 1,6-diphenyl-1,3,5-hexatriene (DPH). The effect of US and Pcs on nucleated erythrocytes morphology was estimated on the basis of microscopic observation.The enhancement of ultrasonically induced membrane damage by both phthalocyanines was observed in case of hemolysis, and membrane surface fluidity, in comparison to ultrasound. The authors also observed changes in the morphology of erythrocytes. The obtained results support the hypothesis that the Pcs enhance ultrasonically induced cell damage in vitro.Furthermore, the influence of ultrasound on phthalocyanines (Pcs) in medium and in cells was tested. The authors observed changes in the phthalocyanines absorption spectra in the medium and the increase in the intensity of phthalocyanines fluorescence in the cells. These data can suggest changes in the structure of phthalocyanines after ultrasound action.     

小鼠近死亡期皮肤损伤活性的FTIR光谱研究

应用傅里叶变换红外(FTIR)光谱分析研究了近死亡期皮肤损伤活性差异。生前5 min组,死后5 min组,正常组的FTIR光谱的峰形、峰高有明显差异：(1)与脂类有关的3 007和1 745 cm-1谱带的峰高,生前伤比死后伤增强。(2)与碳水化合物相关谱带1 160 cm-1的峰高,生前伤比死后伤明显升高。(3)1 640 cm-1谱带为蛋白质相关的酰胺Ⅰ带,损伤组峰高要低于正常组,生前伤中最低。这一结果表明,FTIR光谱分析技术有望成为法医近死亡期皮肤损伤活性判定的有效方法。     

Local defects in the nanostructure of the membrane of erythrocytes upon ionizing radiation of blood

The purpose of the study is to investigate local topological defects in the erythrocyte membranes resulting from the ultraviolet (UV) radiation of blood in vitro. Biological effects in the erythrocytes after exposure to UV radiation at a wavelength of 254 nm are equivalent to those after γ radiation. It has been shown that oxidative processes developing in a suspension upon UV radiation result in the disruption of the nanostructure of the membranes of erythrocytes. In the experiments, typical topological defects in the membrane nanostructure were observed. The parameters of the defects differed from the characteristics of the nanostructure of the control cell membrane without irradiation. The characteristic dimensions of the topological defects are commensurate with the size of the spectrin matrix. As a result of the exposure to the UV radiation, polymorphism of the erythrocytes was observed.     

Secondary-electron SEM bioimaging of human erythrocytes in bloodstains on high-carbon steel substrate without specimen preparation

The imaging of most biological samples via conventional scanning electron microscope (SEM) in secondary-electron mode involves routinely some kind of specimen preparation. Conventional SEMs are still used when a low-vacuum or variable-pressure SEM (usually known as 'environmental', or ESEM) is not available. But that preparatory approach may be undesirable in certain cases, for instance in museum specimens, forensic evidences or clinical samples. This report details a simple, low-cost, and sample-saving bioimaging protocol without specimen preparation, by using removable plastic conducting carbon cement, and then working under ex-profeso SEM conditions, i.e., by using an SEM in secondary-electron mode just like an ESEM. The successful use to image up to high magnifications human erythrocytes in bloodstains on an extensively bloodsmeared, high-carbon steel surgical blade is reported as an example of the potential of this procedure.     

Human bloodstains on bone artefacts: an SEM intra- and inter-sample comparative study using ratite bird tibiotarsus

Apart from their forensic significance in crime investigation, human bloodstains have an anthropological interest due to their occurrence on certain traditional weapons and ritual objects. Previously, a guiding study of erythrocytes in experimental samples including domestic sheep (Ovis aries) tibia was carried out using a scanning electron microscope (SEM). Here, a comparative SEM study to reveal the potential differences in bloodstain surface morphology as a function of intra-sample (smear region) and inter-sample (individual smear, smearing mechanism, bone origin) parameters is reported. A fragment of emu (Dromaius novaehollandiae) tibiotarsus was smeared with an adult man’s peripheral blood. After air-drying and storing indoors, the boundary and neighbouring inner areas of the three individual bloodstains obtained were examined via secondary electrons in a variable-pressure SEM working in low-vacuum mode. As a whole, desiccation microcracks were present, the limits between the smear and the substrate appeared poorly defined, and no erythrocyte negative replicas were observed in the examined areas. In addition, a putative fibrin network, more or less embedded in the dried plasma matrix, was observed in the smears’ boundary. Regarding the smear region in sliding smears, the periphery and boundary revealed to be different, while the head and tail were similar. Considering individual sliding smears, they had similar characteristics. Relating to the smear region as a function of the smearing mechanism, the periphery was different whether sliding or touching, while the boundary was similar in sliding and touching smears. Concerning the smear region as a function of the bone origin, the periphery revealed to be similar in both ratite and mammalian bone, while the boundary did different in ratite and mammalian bone. The results of this study show that SEM examination can be used fruitfully to detect bloodstains on ratite bone. Combined with previous SEM results in domestic sheep bone, they suggest, further, that blood remains can be detected on objects made of bone irrespectively of the mammalian or ratite origin of this raw material.     

A Study on Uptake and Localization of Merocyanine 540 (MC540) in Murine Myeloid Leukemia Cells by Confocal Laser Scanning Microscopy (CLSM)

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A Study on Uptake and Localization of Merocyanine 540 (MC540) in Murine Myeloid Leukemia Cells by Confocal Laser Scanning Microscopy (CLSM)

The intracellular localization of merocyanine 540 (MC540), a photosensitizer commonly used in the photo-inactivation of leukemia cells, was studied using confocal laser scanning microscopy. It was found for the first time that MC540 not only localized in the plasma membrane but also in the cytoplasm and the nuclear membrane of the murine myeloid leukemia M1 and WEHI 3B (JCS) cells. Exposure of MC540 treated leukemia cells to light under conditions that could cause photobleaching did not cause the redistribution of cell-bound MC540. Rapid localization of MC540 in the cytoplasm was observed 5 minutes after exposure of leukemia cell to MC540, indicating that MC540 could promptly be internalized by these two leukemia cell lines. In contrast, localization of MC540 was limited only to the plasma membrane of erythrocytes. These results suggest that the binding pattern of MC540 is cell type dependent and may be related to the efficacy of photosenitization in photodynamic therapy.     

Are the evidences of forensic entomology preserved in ethanol suitable for SEM studies?

In forensic practice, the use of arthropod evidences to estimate the postmortem interval is a very good approach when the elapsed time from death is long, but it requires the correct identification of the specimens. This is a crucial step, not always easy to achieve, in particular when dealing with immature specimens. In this case, scanning electronic microscopy (SEM) can be useful, but the techniques used to preserve specimens in forensic practice are usually different from those used to prepare specimens for SEM studies. To determine whether forensic evidences preserving techniques are also compatible with SEM analysis, we have compared specimens of all the immature stages of Calliphora vicina Robineau-Desvoidy, 1830 (Diptera, Calliphoridae) preserved in 70% ethanol, with others prepared with aldehydic fixative techniques that are more appropriate for SEM studies. At the same time, two drying techniques have also been compared with both fixative techniques, the critical point drying and air-drying following with hexamethyldisilizane treatment (HMDS). Our results indicate that there are not basis against recommending the use of ethanol to preserve forensic entomological evidences and that both drying methods appear to offer good results for second and third instar larvae, although HMDS behaves better with eggs and pupae.     

Fluorescent Methods to Study DNA,RNA, Proteins and Cytoplasmic Membrane Polarization in the Pentachlorophenol-Mineralizing Bacterium <Emphasis Type="Italic">Sphingomonas</Emphasis> sp. UG30 During Nutrient Starvation in Water

The effect of sodium pentachlorophenolate (NaPCP) exposure on the nutrient-starved pentachlorophenol (PCP)-mineralizing bacterium Sphingomonas sp. UG30 was assessed using fluorescent methods to measure DNA, RNA, total cellular protein, and cytoplasmic membrane proteins. UG30 cells were inoculated into sterilized Speed River (Guelph, ON, Canada) water samples in the presence of 50, 100, and 250 ppm NaPCP. No marked changes were observed in the total cellular DNA, RNA or protein levels over 90 d, indicating the macromolecular composition of UG30 was not affected by both nutrient limitation and NaPCP. Total cell counts as determined by DAPI staining also did not change over 90 d. Over the same period, viable counts decreased with increasing concentrations of NaPCP. At 250 ppm NaPCP, viable cell counts decreased over 6 orders of magnitude after 1 hr exposure. Cell numbers partially recovered once NaPCP was degraded. The UG30 cytoplasmic membrane polarization ratio also decreased after NaPCP was depleted. The decreased polarization value at the end of the study period suggested the UG30 membrane was more fluid and that this increase in fluidity was due to nutrient starvation effects rather than exposure to NaPCP. These results indicated that UG30 is a robust organism that is able to degrade NaPCP even under adverse conditions and fluorescent methods are useful for determining macromolecular concentrations and cytoplasmic membrane polarization values.     

Alteration of diffusion tensor parameters in postmortem brain

In autopsy of humans, there is usually an interval of hours to days between death and tissue fixation, during which the cadaver is stored below room temperature to retard tissue autolysis. We have attempted to model this process and evaluate the alteration in diffusion indices of the postmortem brain in pigs, which were kept at 4°C. The pigs were scanned prior to death and at 3, 6, 9, 12, 18, 24, 30, 36, 42, 48 and 72 h postmortem. Regions of interest were placed in the corpus callosum, internal capsule, periventricular and subcortical white matter anteriorly and posteriorly. There was a slight increase in fractional anisotropy (FA) in the first 3 h postmortem. The FA remained stable up to 72 h postmortem. There was a marked decrease in trace, eigenmajor (λmajor), eigenmedium (λmedium) and eigenminor (λminor), particularly in the first 3 h following death. This study supports the utility of measuring diffusion anisotropy if the time elapsed between death and tissue fixation is within 3 days. However, trace and eigenvalues decreased markedly within the first few hours postmortem. Therefore trace and eigenvalues obtained from ex vivo studies cannot be extrapolated to in vivo studies.     

Structural and electrical properties of tracks of heavy ions in polyethylene terephthalate and mica

An image of the entrance of high-energy ions of krypton, xenon, and bismuth on the surface of polyethylene terephthalate (PET) and mica is obtained. It is shown that in both cases the tracks have the appearance of protuberances. The formation of protuberances is associated with the destruction of material in the region of the track, the formation of amorphous structure, and the removal of low-molecular-weight radiolysis products as gaseous substances. Storage of the irradiated PET samples in water and a weak alkaline solution for several hours changes the surface topography due to hydrolysis of the material and the removal of radiolysis products into the solution. The kinetics of etching tracks in a weak alkaline solution and the formation of pores are studied. The stream function, electrical resistance, and permeability of the tracks in KCl, LiCl, MgCl₂, and BaCl₂ electrolytes are measured. The surface charge of the tracks is calculated from these values.     

C2C12 myoblast sensitivity to different apoptotic chemical triggers

Apoptosis is a form of cell death crucial for normal development and tissue homeostasis. Its typical features include chromatin changes, nuclear breakdown, plasma membrane blebbing and splitting of cellular content into apoptotic bodies, that progressively undergo phagocytosis.Apoptosis is considered essential for skeletal muscle development, where defective cells are deleted during differentiation. In addition, it plays a relevant role in several muscle myopathies, as well as in denervation and disuse.The aim of this study was to evaluate muscle cell sensitivity to different apoptotic triggers, acting through different mechanisms of action. Chemical agents, active against distinct intracellular targets, such as mitochondrial respiratory chain and DNA, have been chosen to better highlight cell death mechanisms. To induce apoptosis, C2C12 myoblasts have been exposed to H₂O₂, staurosporine, cisplatin and etoposide, at different doses and incubation times, and they have been analysed by flow cytometry, scanning and transmission electron microscopy.Flow cytometry analysis revealed a certain subdiploid peak after all treatments. The best apoptotic effect was observable, as confirmed at reverted microscope, at minimum doses and after the major exposure time.At ultrastructural level programmed cell death has been observed. Characteristic chromatin condensation and margination, as well as apoptotic bodies, frequently appeared, even if in the presence of secondary necrosis; surface blebs were also observed during scanning microscopic observation.In particular, exposure to H₂O₂ or staurosporine showed the largest number of myoblasts in late apoptotic stages and in secondary necrosis. Cisplatin treatments revealed few early apoptotic cells. The analysis of etoposide-induced apoptosis was in agreement with data obtained from flow cytometry, indicating a significant increase of apoptotic cell number.These results suggest that all conditions are able to induce apoptosis in C2C12 myoblasts, which occurs, considering trigger mechanisms of action, mostly following the mitochondrial pathway, if not excluding that due to DNA damage. Therefore, mitochondria permeability alteration is an important step in skeletal muscle programmed cell death. This last conclusion seems to have a significant relevance in understanding the mechanisms involved in muscle disorders, denervation and chronic muscle disuse, conditions frequently characterized by a decline in mitochondrial content and by an increase of mitochondrial apoptosis susceptibility.     

大鼠死后肺脏FTIR光谱变化与死亡时间的关系

应用傅里叶变换红外(FTIR)光谱分析大鼠死后肺脏组织随死亡时间推移的化学降解过程,为法医死亡时间推断提供新的研究方法。结果表明,随着死亡时间的推移,大鼠肺脏组织FTIR光谱的主要吸收峰峰位没有明显变化,而其峰高有明显差异：(1)与核酸有关的1080,1241cm-1谱带的峰强呈下降趋势。(2)酰胺Ⅰ(I₁₆₄₇),Ⅱ(I₁₅₄₁)峰强比随死亡时间推移下降。1338和1313cm-1处的吸收峰强略有升高。(3)1460, 1400,1170 cm-1的谱带没有明显变化趋势。(4)死亡72 h后,1120 cm-1处出现新的吸收峰,吸收强度随时间推移逐渐增高;这一结果表明FTIR光谱分析技术有望成为法医死亡时间推断的有效方法。     

Radiation response of cell organelles

The cellular responses to various form of radiation, including ionizing- and UV-irradiation or exposure to electromagnetic fields is manifested as irreversible and reversible structural and functional changes to cells and cell organelles. Moreover, beside the morphological signs related to cell death, there are several reversible alterations in the structure of different cell organelles. The radiation-induced changes in the supramolecular organization of the membranes, including plasma membrane, and different cell organelle membranes, play a significant role in the development of acute radiation injury. These signs of radiation-induced reversible perturbation biological membranes reflect changes in the organization and/or composition of the glycocalix, modified activity and/or distribution of different membrane domains, including enzymes and binding sites. The observed changes of the cell surface micromorphology and the alteration of intercellular connections are closely related to the reorganization of the cytoskeletal elements in the irradiated cells. The mitochondria, endoplasmic reticulum, Golgi-complex, the lysosomal system have long been considered to be direct intracellular targets of irradiation. The listed morphological alterations of nuclear chromatin (e.g. changes of fine structure, altered number of nucleolar organizing regions and micronuclei, development of chromosome aberrations) may originate from the radiation-induced damage to the supramolecular organization of DNA and/or nucleus specific proteins. These endpoints of radiation effects resulted as direct consequence(s) of absorbed radiation energy, and indirectly altered intra-, intercellular communication or modified signal transduction. Some complementary data suggest that all these effects are not strictly specific to radiation and may be best considered as general stress responses, similar to those observed after application of various injurious agents and treatments to cells. Moreover, they may be equally responsible for direct degradation of supramolecular component of cells, altered signal transduction, or changes in the amount or ratio of any extracellular mediators upon irradiation. Nevertheless, qualitative and/or quantitative evaluation of any changes of chromosomes by different techniques (morphological analysis of metaphase chromosomes, fluorescent in situ hybridization, development of micronuclei etc.) are useful biological indicators as well as "biological dosimeters" of radiation injury. It is suggested, that some modern methods such as immunohistochemical detection of different proteins, specific markers of cell organelles and cytoskeleton, inspection of distribution of cell surface charged sites and different membrane domains and application of tracer substances may all be included into protocols for evaluation of cell alterations induced by different types and intensities of radiation.     

Sensitizer localization and immune response in photodynamic therapy of B16 cells

This paper proposes to extend the exploration of mouse melanoma B16 cells death by photodynamic therapy (PDT), under irradiation with different light sources and in the presence of 5,10,15,20-tetrap-sulphonato-phenyl-porphyrin (TSPP). The viability studies showed that B16 mouse melanoma is sensitive to photodynamic damage induced by TSPP 1 mM for either one, two, three or four hours. The control had TSPP added immediately prior to timelapse imaging (no incubation). They were then irradiated with red light He-Ne laser (λ = 632.8 nm, energy 180 J/cm² for 20 min). Also, it has been used a laser diode GaInAs 25 mW/cm², λ = 650 nm. The cells demonstrated clear morphological changes associated with apoptosis by mitochondrial pathway. There were changes in texture, as expected. Changes appeared to occur more quickly at lamp irradiation than at HeNe and GaInAs diode laser. Addition of TSPP just prior to exposure and observation, with no incubation, did not result in changes in cell morphology or cell death. Also, the proteins changes have been observed, because those with high molecular weights have been scissored under irradiation and this could be reason of the proteins concentrating in the area of low molecular weights, and the dissapearing of the proteic band of 75 kDa in the electrophoregramm. The immunized animals with B16-TSPP had the highest survival rate (40 days) by comparison with the control (death at 20 days) or with immunized animals with supernatants B16 (death at 25 days).     

pH协同血清白蛋白对单个活态人红细胞作用的拉曼光谱研究

红细胞是维持生命最重要的细胞之一,pH是影响其结构功能的重要因素。 结合人血液中存在血清白蛋白,且可能会对红细胞起作用的特点,在模拟人体内血液中血清白蛋白与血红蛋白摩尔比值的条件下,研究pH协同血清白蛋白对单个活态人红细胞的形态,细胞膜变形能力及胞内血红蛋白结构变化的影响。 结果显示,不同pH下,红细胞的表面形态、 膜变形能力的变化规律与其胞内血红蛋白结构的变化有关：在pH 4.14,4.76,10.18时,红细胞胞内血红蛋白螺旋结构的减少、 疏水性氨基酸的暴露,血红素珠蛋白结合状态的改变,影响了血红蛋白结构的稳定性,从而导致红细胞的结构发生变化,其形态、 膜变形能力与正常值相比,变化结果显著。 而在pH 6.51和7.80下,血红蛋白的拉曼谱结果并未发现有上述变化情况,胞内血红蛋白的结构没有发生改变,红细胞的形态、 膜变形能力也接近于正常值。 在pH 5.49和8.76下,红细胞的表面形态和膜变形能力都有不同程度的变差,但其胞内血红蛋白的结构并没有改变,提示红细胞的形态和变形能力的改变可能是可逆的。 结果表明,pH协同血清白蛋白的作用下,血清白蛋白能对红细胞起保护作用,增强红细胞调节、 缓冲环境pH变化的能力。 研究结果全面地揭示了在模拟人体内血液中血清白蛋白与血红蛋白摩尔比值的情况下,红细胞结构功能的变化情况,不仅有助于阐明红细胞在有关生理、 病理状态下的结构与功能的变化,而且对其临床表现与有关防治对策都有重要的指导意义。     

The effect of air exposure on palladium–copper composite membranes

It was found that when electrolessly deposited thin Pd and Pd–Cu membranes were exposed to air at temperatures above 350 °C, their H₂ flux increased substantially immediately after the air exposure, then decreased to a new steady-state value. While this was a quasi-reversible change for the H₂ flux, the flux of insoluble species, such as N₂, irreversibly increased with every air exposure but by a much smaller extent. The extent of these changes was found to be dependent on the exposure time and the temperature of the tests. Thus, we decided to investigate the effect of gas exposures on the properties of these materials.

Palladium and palladium–copper films, prepared by electroless deposition on ceramic supports, and commercial foils were exposed to air, hydrogen and helium at 500 and 900 °C for times varying from 1 h to 1 week with the objective of determining the effect of the different exposure conditions on the surface morphology, the flux of different penetrants and the crystalline structure of the materials. Atomic force microscopy (AFM) and X-ray diffraction (XRD) were used to study the changes occurring in the films under those conditions.

It was observed that the exposure of both the electroless films and the foils to hydrogen and air markedly modified their surface morphology. The hydrogen exposure tended to smooth the surface features whereas the oxygen exposure created new surface features such holes and large peaks. Additionally it was found that the air exposure produced some oxidation of the film to create PdO.

These results suggested that a common hypothesis stating that air oxidation just cleans the surface of the membrane might not be sufficient to explain all of those changes. A contributing effect of air exposure may be the increase in surface area due to the formation of palladium oxide. However, the extent of the surface area increase was insufficient to explain the increase in steady-state H₂ flux.     

Noise-induced threshold shift and cochlear pathology in the Mongolian gerbil

Groups of six mongolian gerbils were exposed to two-octave (1414-5656 Hz) band noise for 1 h at 100, 110, and 120 dB SPL. Threshold shift at several frequencies was measured 0.5, 3, 6, and 12 h, and 1-28 days after exposure. Final thresholds were determined at least two months postexposure. Extensive threshold shift was observed in all groups 0.5 h after exposure (TS0.5h). Where threshold shift increased in the initial hours after exposure, such increases were correlated with eventual permanent threshold shift (PTS). Recovery of thresholds from 1-28 days after exposure was approximately exponential, and slowest at the edges of the exposure band. PTS was seen in the 110 and 120 dB SPL groups. With TS0.5h of 50 dB or less, no PTS resulted. With TS0.5h above 50-60 dB, eventual PTS increased linearly with a slope of about 1.25 PTS/TS0.5h. Cochlear damage was evaluated by light microscopy. The relationship between hair cell loss and PTS was consistent with an inner hair cell threshold about 40 dB higher than that of outer hair cells. It is suggested that recovery from noise-induced threshold shift may involve different mechanisms in the two types of hair cells.