Comparison of PET imaging with 64Cu-liposomes and 18F-FDG in the 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch model of oral dysplasia and squamous cell carcinoma.

PURPOSE: Currently, 2-deoxy-2-[(18)F]fluoro-D-glucose ((18)F-FDG) is the gold standard radiotracer for staging of head and neck cancer; however, the low sensitivity of this tracer can impede detection of early lesions. (64)Cu-liposomes accumulate in various cancers and provide both a sensitive tracer and an indication of the biodistribution of nanotherapeutics. Here, the accumulation of (64)Cu-liposomes in early and established cancers is assessed and compared with (18)F-FDG in a head and neck cancer model. METHODS: Lesions ranging from mild dysplasia to squamous cell carcinoma were induced in a hamster model of head and neck cancer by topical application of 7,12-dimethylbenz[a]anthracene to the buccal pouch. The hamsters were imaged with micro-positron emission tomography using (18)F-FDG and (64)Cu-liposomes. RESULTS: At 24 h postinjection, (64)Cu-liposome accumulation exceeded the accumulation of (18)F-FDG in every pathologic grade. The lesion-to-cheek pouch (background) ratio and lesion-to-brain ratio were also higher for (64)Cu-liposomes than for (18)F-FDG. CONCLUSION: Imaging of a nanotracer such as (64)Cu-liposomes can improve the visualization of head and neck tumors. Accumulation of liposomal particles in head and neck tumors over various pathologic grades averaged 3.5%ID/cc demonstrating the potential for liposomal therapy with targeted chemotherapeutic agents.

Multimodal in vivo imaging of oral cancer using fluorescence lifetime, photoacoustic and ultrasound techniques.

This work reports a multimodal system for label-free tissue diagnosis combining fluorescence lifetime imaging (FLIm), ultrasound backscatter microscopy (UBM), and photoacoustic imaging (PAI). This system provides complementary biochemical, structural and functional features allowing for enhanced in vivo detection of oral carcinoma. Results from a hamster oral carcinoma model (normal, precancer and carcinoma) are presented demonstrating the ability of FLIm to delineate biochemical composition at the tissue surface, UBM and related radiofrequency parameters to identify disruptions in the tissue microarchitecture and PAI to map optical absorption associated with specific tissue morphology and physiology.

In vivo validation of a bimodal technique combining time-resolved fluorescence spectroscopy and ultrasonic backscatter microscopy for diagnosis of oral carcinoma.

Tissue diagnostic features generated by a bimodal technique integrating scanning time-resolved fluorescence spectroscopy (TRFS) and ultrasonic backscatter microscopy (UBM) are investigated in an in vivo hamster oral carcinoma model. Tissue fluorescence is excited by a pulsed nitrogen laser and spectrally and temporally resolved using a set of filters/dichroic mirrors and a fast digitizer, respectively. A 41-MHz focused transducer (37-µm axial, 65-µm lateral resolution) is used for UBM scanning. Representative lesions of the different stages of carcinogenesis show that fluorescence characteristics complement ultrasonic features, and both correlate with histological findings. These results demonstrate that TRFS-UBM provide a wealth of co-registered, complementary data concerning tissue composition and structure as it relates to disease status. The direct co-registration of the TRFS data (sensitive to surface molecular changes) with the UBM data (sensitive to cross-sectional structural changes and depth of tumor invasion) is expected to play an important role in pre-operative diagnosis and intra-operative determination of tumor margins.

Artificial muscle for reanimation of the paralyzed face: durability and biocompatibility in a gerbil model.

BACKGROUND: Current management of permanent facial paralysis centers on nerve grafting and muscle transfer; however, limitations of those procedures call for other options. OBJECTIVES: To determine the durability and biocompatibility of implanted artificial muscle in a gerbil model and the degree of inflammation and fibrosis at the host tissue-artificial muscle interface. METHODS: Electroactive polymer artificial muscle (EPAM) devices engineered in medical-grade silicone were implanted subcutaneously in 13 gerbils. The implanted units were stimulated with 1 kV at 1 Hz, 24 h/d via a function generator. Electrical signal input/output was recorded up to 40 days after implantation. The animals were euthanized between 23 and 65 days after implantation, and the host tissue-implant interface was evaluated histologically. RESULTS: The animals tolerated implantation of the EPAM devices well, with no perioperative deaths. The muscle devices created motion for a mean of 30.3 days (range, 19-40 days), with a mean of 2.6 × 106 cycles (range, 1.6 × 106 to 3.5 × 106 cycles). Histologic examination of the explanted devices revealed the development of a minimal fibrous capsule surrounding the implants, with no evidence of bacterial infection or inflammatory infiltrate. No evidence of device compromise, corrosion, or silicone breakdown was noted. CONCLUSIONS: Artificial muscle implanted in this short-term animal model was safe and functional in this preliminary study. We believe that EPAM devices will be a safe and viable option for restoration of facial motions in patients with irreversible facial paralysis.

Comparison of effectiveness of silicone gel sheeting with microporous paper tape in the prevention of hypertrophic scarring in a rabbit model.

OBJECTIVE: To determine the effectiveness of treating scars with microporous paper tape or silicone gel sheeting (SGS) in preventing hypertrophic scarring. METHODS: Forty hypertrophic scars were induced in a validated rabbit ear model. Wounds were randomized and bandaged for 30 days with either SGS (20 wounds), paper tape (20 wounds), or untreated controls (40 wounds). Two outcome measures of hypertrophic scarring included (1) histologic measurement of scar elevation index (SEI) and (2) blinded photograph analysis using a visual analog scale. RESULTS: In histologic comparison, no difference in mean (SE) SEI between treatment groups was seen (paper tape group, 1.32 [0.2]; SGS group, 1.41 [0.18]; control, 1.35 [0.23]; P = .51). In photographic analysis, both treatment groups were superior to the control group (P < .01), whereas no difference was seen between the SGS and paper tape groups (P = .88). CONCLUSIONS: Paper tape and SGS demonstrated equal effectiveness in the prevention of hypertrophic scarring on visual analysis, whereas histologic analysis demonstrated no difference in treatment groups from controls. The effectiveness of paper tape in preventing hypertrophic scarring in humans will require further laboratory and clinical investigation.

Fluorescence lifetime imaging microscopy: in vivo application to diagnosis of oral carcinoma.

A compact clinically compatible fluorescence lifetime imaging microscopy (FLIM) system was designed and built for intraoperative disease diagnosis and validated in vivo in a hamster oral carcinogenesis model. This apparatus allows for the remote image collection via a flexible imaging probe consisting of a gradient index objective lens and a fiber bundle. Tissue autofluorescence (337 nm excitation) was imaged using an intensified CCD with a gate width down to 0.2 ns. We demonstrate a significant contrast in fluorescence lifetime between tumor (1.77+/-0.26 ns) and normal (2.50+/-0.36 ns) tissues at 450 nm and an over 80% intensity decrease at 390 nm emission in tumor versus normal areas. The time-resolved images were minimally affected by tissue morphology, endogenous absorbers, and illumination. These results demonstrate the potential of FLIM as an intraoperative diagnostic technique.

Long-term hearing loss in gerbils with bacterial meningitis treated with superoxide dismutase.

HYPOTHESIS: The treatment of superoxide dismutase (SOD) in gerbils with bacterial meningitis will not only prevent cochlear fibrosis and neo-ossification but also reduce hearing loss. BACKGROUND: SOD an O2-scavenger, has been shown to prevent cochlear fibrosis and neo-ossification in gerbils infected with bacterial meningitis when injected intrathecally. The objective of this study is to investigate the effects of SOD on long-term hearing loss in gerbils infected with bacterial meningitis and to assess the relationship between hearing results and the amount of fibrosis. The effectiveness of middle ear infusion of SOD will also be examined. METHODS: Meningitis was induced in 3 groups of 10 gerbils with injection of Streptococcus pneumoniae into the cisterna magna. Group 1 received intrathecal SOD, group 2 received a middle ear infusion of SOD, and group 3, the control group, received no SOD. Histologic data and auditory brainstem responses were obtained from each gerbil. RESULTS: In the intrathecal SOD group, the average deterioration in pure tone thresholds between the preoperative baseline and 15 weeks after induction of meningitis at 4, 8, 16, and 32 kHz was significantly less than that of the middle ear SOD and the control group (p = 0.001). There was no significant difference between the middle ear SOD and the control group. There was no fibrosis in the intrathecal SOD group, 15% of the gerbils developed an average of 11% fibrosis in the middle ear SOD group, and 20% of the gerbils developed an average of 15% in the control group. CONCLUSION: Intrathecal infusion of SOD not only prevented cochlear fibrosis and neo-ossification after bacterial meningitis but also decreased subsequent hearing loss.

Gerbilline cholesteatoma development Part III. Increased proliferation index of basal keratinocytes of the tympanic membrane and external ear canal.

OBJECTIVE: To quantify the rate of basal cell division for keratinizing epithelium (KE) of the tympanic membrane (TM) and external ear canal (EAC) in spontaneous and induced gerbilline cholesteatomas. STUDY DESIGN AND SETTING: Cholesteatomas (3 spontaneous and 5 by induction) were labeled with tritiated thymidine for autoradiography and a KE proliferation index (PI) was determined. The PI was defined as the average number of labeled cells/mm overall and per anatomic region. RESULTS: For all regions combined, the PI was 27.3 in ears with cholesteatoma and 4.1 in normal ears (P < 0.0001). Additionally, there were significant regional differences in the PI in both normal ears and ears with cholesteatoma. CONCLUSION: The KE of cholesteatomas in gerbils proliferates at approximately 7 times the rate measured in control ears. SIGNIFICANCE: Hyperproliferation of keratinocytes is a causative factor in the development and progression of spontaneous and experimental cholesteatomas in gerbils.

Gerbilline cholesteatoma development Part I: Epithelial migration pattern and rate on the gerbil tympanic membrane: comparisons with human and guinea pig.

OBJECTIVE: To determine the migration rate and pattern for keratin on the tympanic membrane (TM) of the gerbil and guinea pig in comparison to human data and determine which species is an appropriate model for investigating the relationship of epithelial clearance to cholesteatoma formation. STUDY DESIGN AND SETTING: Ink drops were placed on the TM and their locations plotted daily. RESULTS: Gerbils demonstrated a radial migration pattern away from the umbo, identical to that reported for humans, although the rate was 0.32 mm/day-3 times the human rate of 0.1 mm/day. Guinea pigs were significantly different from gerbils and humans, with a rate of 0.79 mm/day and a pattern of superior/inferior migration without anterior-posterior movement. CONCLUSION: Gerbils more closely resemble humans in rate and pattern of epithelial migration. SIGNIFICANCE: Gerbils represent the most appropriate model for determining the relationship between keratin migration and cholesteatoma formation.

Gerbilline cholesteatoma development. Part II: temporal histopathologic changes in the tympanic membrane and middle ear.

OBJECTIVE: Determine the sequence of gross and histopathologic change to the normal middle ear (ME), tympanic membrane (TM), and external auditory canal (EAC) during spontaneous gerbilline cholesteatoma development. STUDY DESIGN AND SETTING: Sixty-six gerbils were examined weekly and periodically sacrificed for analysis. RESULTS: Cholesteatoma development followed this sequence: 1) slightly thickened pars flaccida (PF) without ME effusion, 2) thickened PF with ME effusion, 3) continuous buildup of EAC debris, and 4) complete occlusion of the lateral EAC. In the cholesteatoma group, keratinizing epithelium (KE) thickness was increased in all regions over normal controls and regional gradations in the TM remained the same. CONCLUSION: Spontaneous cholesteatoma formation was significantly associated with middle ear effusion, a failure to clear canal debris with resultant retrograde thickening of the EAC, PF, and then pars tensa. SIGNIFICANCE: Inflammatory insult and alteration of the epithelial clearance mechanism appears to result in spontaneous cholesteatoma formation in gerbils as hypothesized for humans.

Nonhuman primate models of intrauterine cytomegalovirus infection.

Congenital human cytomegalovirus (HCMV) infection has long been recognized as a threat to the developing fetus, even though studies have shown that only a subset of congenital infections results in clinical signs of disease. Among the estimated 8000 children who develop sequelae from congenital CMV infection each year in the United States alone, most suffer permanent developmental defects within the central nervous system. Because there is currently no approved vaccine for HCMV, and anti-HCMV drugs are not administered to gravid women with congenital infection because of potential toxicity to the fetus, there is a clear clinical need for effective strategies that minimize infection in the mother, transplacental transmission of the virus, and/or fetal disease. Animal models provide a method to understand the mechanisms of HCMV persistence and pathogenesis, and allow for testing of novel strategies that limit prenatal infection and disease. The rhesus macaque model is especially well suited for these tasks because monkeys and humans share strong developmental, immunological, anatomical, and biochemical similarities due to their close phylogenetic relationship. This nonhuman primate model provides an invaluable system to accelerate the clinical development of promising new therapies for the treatment of human disease. This review addresses salient findings with the macaque model as they relate to HCMV infection and potential avenues of discovery, including studies of intrauterine CMV infection. The complexity of the natural history of HCMV is discussed, along with the ethical and logistical issues associated with studies during pregnancy, the recent contributions of animal research in this field of study, and future prospects for increasing our understanding of immunity against HCMV disease.

Role of tumor necrosis factor-alpha in sensorineural hearing loss after bacterial meningitis.

HYPOTHESIS: Blockade of tumor necrosis factor-alpha with tumor necrosis factor-alpha antibody will reduce the extent of cochlear injury and hearing loss associated with Streptococcus pneumoniae meningitis. BACKGROUND: Inflammatory mediators play a significant role in the morbidity associated with bacterial meningitis, including hearing loss and labyrinthitis ossificans. Previous studies have shown the attenuation of hearing loss by the nonspecific blockade of such pathways. METHODS: Fifty Mongolian gerbils were divided into four groups. Auditory brainstem response testing was conducted to measure hearing thresholds. Streptococcus pneumoniae meningitis was induced in Groups 1 and 2. Group 2 was then given a single intraperitoneal injection of tumor necrosis factor-alpha antibody, whereas Group 1 received phosphate-buffered saline. Uninfected animals in Groups 3 and 4 were implanted with osmotic pumps that delivered a continuous 8-day intrathecal flow of either tumor necrosis factor-alpha (Group 4) or phosphate-buffered saline (Group 3). After 6 weeks, auditory brainstem response testing was repeated. The cochleas were harvested and analyzed histomorphometrically. RESULTS: Group 2 animals with Streptococcus pneumoniae meningitis that also received tumor necrosis factor-alpha antibody developed significantly less hearing loss than Group 1 animals with meningitis alone. The decrease in the average threshold at 4, 8, 16, and 32 kHz was 31, 30, 25, and 28 dB sound pressure level, respectively (p < 0.0092 for each). Furthermore, histomorphometric analysis showed significantly less damage to the organ of Corti, spiral ganglion, spiral ligament, and stria vascularis in Group 2. Conversely, tumor necrosis factor-alpha induced meningitis animals (Group 3) showed increased hearing loss compared with phosphate-buffered saline controls (Group 4), with p < 0.0001 at all frequencies. CONCLUSION: Tumor necrosis factor-alpha plays an important role in cochlear injury after bacterial meningitis. Blockade of tumor necrosis factor-alpha reduces postmeningitic hearing loss and cochlear injury. Induction of meningitis with intrathecal tumor necrosis factor-alpha also resulted in hearing loss and cochlear injury similar to bacterial meningitis.

Fine structure histopathology of labyrinthitis ossificans in the gerbil model.

Labyrinthitis ossificans (LO) is the pathological deposition of new bone within the lumen of the cochlea and labyrinth. This process occurs most commonly as a result of infection or inflammation affecting the otic capsule. Trauma and vascular compromise can also lead to neo-ossification within the otic capsule. The mechanism that regulates this process remains unestablished. This study details the end-stage histopathology in high-resolution plastic thin sections. Twenty Mongolian gerbils were infected by intrathecal injection of Streptococcus pneumoniae type 3 followed by subcutaneous penicillin G procaine (8 days) and were painlessly sacrificed 3 months later. The cochleas were serially divided and sectioned for light and electron microscopy. Sixteen of 20 animals (27 of 40 cochleas) demonstrated LO. Cochlear damage was most extensive in the vestibule and basal turn and decreased toward the apex, which often appeared normal. The histopathologic findings consisted of 1) new bone, calcospherites, osteoid, and fibrosis without dense connective tissue or osteoblasts extending from the endosteal wall into the lumen of the vestibule and scala tympani; 2) areas of dense connective tissue and osteoid enclosed by epithelial cells conjoined with the organ of Corti, stria vascularis, spiral ligament, and vestibular (Reissner's) membrane; and 3) partial to complete loss of the organ of Corti, spiral ligament cell bodies, stria vascularis, and spiral ganglion cells. Osteoblastic activity was not demonstrated in end-stage ossification in LO in the gerbil model. Neo-ossification appears to occur by calcospherite deposition along collagen-like fibrils within osteoid. The destruction of the organ of Corti, spiral ganglion cells, stria vascularis, and cells of Reissner's membrane and the spiral ligament occurs even in the absence of ossification of the cochlear duct.

The effects of superoxide dismutase in gerbils with bacterial meningitis.

BACKGROUND: Inflammatory products, such as oxygen radicals generated during the course of bacterial meningitis, can damage nerve endings, hair cells, and/or supporting cells in the cochlea. Superoxide dismutase (SOD), an O2-scavenger, has been shown to play an important role in the protection against radical toxicity in various animal experiments. OBJECTIVE: To study the antioxidant effects of SOD on the inflammatory response of gerbils with bacterial meningitis. STUDY DESIGN: Meningitis was induced in three groups of 10 gerbils by intrathecal (IT) injection of Streptococcus pneumoniae into the cisterna magna. Group 1 received IT SOD, group 2 received intramuscular (IM) SOD, and group 3, the control group, received IM normal saline. Histologic data and auditory brainstem responses (ABR) were obtained from each gerbil. RESULTS: Fibrosis and/or neo-ossification were near absent in the IT SOD group and significantly less fibrosis occurred in the IM group (IT vs. IM: P = 0.010; IT vs. control group: P = 0.001). The amount of surviving spiral ganglion cells correlated inversely with the extent of fibrosis (r = -0.753, P < 0.00001). CONCLUSIONS: IT injection of SOD significantly reduced cochlear fibrosis and neo-ossification, reduced the spiral ganglion cell loss, and decreased damage of the cochlear components following bacterial meningitis.

Location and timing of initial osteoid deposition in postmeningitic labyrinthitis ossificans determined by multiple fluorescent labels.

OBJECTIVES/HYPOTHESIS: Variable amounts of fibrosis and neo-ossification fill the cochlea following bacterial meningitis. The purpose of the study was to delineate the timing and location of initial ossification following pneumococcal meningitis, as well as subsequent remodeling and resorption, over the 3-month period after infection. STUDY DESIGN: Randomized, double-blind study. METHODS: Fluorochromes are compounds that specifically incorporate into ossifying bone. Sequential addition of different colored fluorochromes during osteoneogenesis define the timing and location of osteoid deposition and mineralization. Mongolian gerbils were infected by intrathecal injection of Streptococcus pneumoniae type 3, and control gerbils received saline. Both groups were injected with calcein on postoperative day 3, followed by xylenol orange, oxytetracycline, and alizarin red on days 7, 14, and 28 respectively. Ten experimental gerbils were killed 24 hours after each label, and an additional group at 84 days after infection. Two groups of 10 control gerbils were killed at 29 and 84 days after treatment. The temporal bones and tibias were harvested, embedded in plastic, and sliced with a diamond saw. Wafers at a thickness of 200 microm were mounted in sequence and examined. RESULTS: Sixteen of 49 experimental animals (33%) were positive for at least one of the fluorescent labels. Fluorescent labeled osteoid was present at all sampling times. Label extended from the endosteal wall into the lumen of the scala tympani between the vestibule and the round window membrane. Discrete sites of fluorescence varied among specimens and were associated with the opening of the cochlear aqueduct, the scala tympani, organ of Corti, and the stria vascularis and spiral ligament in all turns from base to apex. CONCLUSION: The results indicate that osteoid is deposited and begins mineralization by day 3 after infection, at least, and continues, at least, through the first 28 days after infection. There was no apparent resorption of new bone and remodeling by 84 days after infection.

Increased proliferation and migration of epithelium in advancing experimental cholesteatomas.

HYPOTHESIS: Hyperproliferative and migratory process of keratinocytes are part of the pathogenesis of cholesteatoma. BACKGROUND: Cytokeratin (CK) changes were prominent in the most rapidly expanding regions of cholesteatoma formation. METHODS: The three types of animal model-canal ligation (CL), retraction pocket (RP), and propylene glycol (PG)-were induced in Mongolian gerbils. The monoclonal antibodies to CK1/10, CK5/6, and CK13/16 were used for immunohistochemistry. The intensity of immunostaining in the pars tensa of the tympanic membrane was measured using the densitometry and compared with respect to the stage of cholesteatoma and the type of animal model. RESULTS: With cholesteatoma formation, CK expressions were significantly increased at the peripheral part of the pars tensa, the expanding part of cholesteatoma. Among the CKs tested, the prominent changes were observed in expression of CK13/16, a marker for hyperproliferation. Among the animal models, CK changes of CK5/6 and CK1/10 were most prominent in the CL type, whereas those of CK13/16 were more persistent in the RP type. CONCLUSION: These results suggested that complex alterations of epidermal keratinocytes occur during cholesteatoma formation and that hyperproliferative and migratory processes play important roles in the pathogenesis of cholesteatoma.

Expression patterns of cytokeratins in cholesteatomas: evidence of increased migration and proliferation.

Aural cholesteatoma is characterized by invading squamous epithelia with altered growth properties. Cytokeratin (CK) expression is affected in epidermal proliferative diseases and represents the alterations of keratinocyte proliferation, differentiation, and migration. In the present study, the intensity of CK immuno-expression was determined, using densitometry at various sites in experimental cholesteatoma in order to characterize changes of keratinocytes. With cholesteatoma formation, CK4, a marker for non-keratinizing epithelia, increased in the suprabasal layers of the annular external auditory canal (EAC) and at the pars tensa indicating an altered differentiation and migration of keratinocytes. CK5/6, a marker of keratinizing squamous epithelium, increased only at the pars tensa of the tympanic membrane, indicating basal keratinocyte hyperplasia. CK1/10 increased in the suprabasal layer at the annular EAC, and at the peripheral pars tensa, indicating increased terminal differentiation of keratinocytes. CK13/16, markers of differentiation and hyperproliferation, increased in suprabasal layer of the EAC, and at the peripheral pars tensa. However, it decreased in the basal layer of the EAC, indicating hyperproliferation and migration of keratinocytes. The findings of this study support the basal cell hyperplasia hypotheses for the pathogenesis of aural cholesteatoma, with regard to hyperproliferation, migration, and an altered differentiation of keratinocytes.