A Subcutaneous Implant of Tenofovir Alafenamide Fumarate Causes Local Inflammation and Tissue Necrosis in Rabbits and Macaques.

We describe the in vitro and in vivo evaluation of a subcutaneous reservoir implant delivering tenofovir alafenamide hemifumarate (TAF) for the prevention of HIV infection. These long-acting reservoir implants were able to deliver antiretroviral drug for over 90 days in vitro and in vivo We evaluated the implants for implantation site histopathology and pharmacokinetics in plasma and tissues for up to 12 weeks in New Zealand White rabbit and rhesus macaque models. A dose-ranging study in rabbits demonstrated dose-dependent pharmacokinetics and local inflammation up to severe necrosis around the active implants. The matched placebos showed normal wound healing and fibrous tissue encapsulation of the implant. We designed a second implant with a lower release rate and flux of TAF and achieved a median cellular level of tenofovir diphosphate of 42 fmol per 106 rhesus macaque peripheral blood mononuclear cells at a TAF dose of 10 µg/kg/day. This dose and flux of TAF also resulted in adverse local inflammation and necrosis near the implant in rhesus macaques. The level of inflammation in the primates was markedly lower in the placebo group than in the active-implant group. The histological inflammatory response to the TAF implant at 4 and 12 weeks in primates was graded as a severe reaction. Thus, while we were able to achieve a sustained target dose, we observed an unacceptable inflammatory response locally at the implant tissue interface.

Reflex wind-up in early chronic spinal injury: plasticity of motor outputs.

In this study we evaluate temporal summation (wind-up) of reflexes in select distal and proximal hindlimb muscles in response to repeated stimuli of the distal tibial or superficial peroneal nerves in cats 1 mo after complete spinal transection. This report is a continuation of our previous paper on reflex wind-up in the intact and acutely spinalized cat. To evaluate reflex wind-up in both studies, we recorded electromyographic signals from the following left hindlimb muscles: lateral gastrocnemius (LG), tibialis anterior (TA), semitendinosus (ST), and sartorius (Srt), in response to 10 electrical pulses to the tibial or superficial peroneal nerves. Two distinct components of the reflex responses were considered, a short-latency compound action potential (CAP) and a longer duration bout of sustained activity (SA). These two response types were shown to be differentially modified by acute spinal injury in our previous work (Frigon A, Johnson MD, Heckman CJ. J Physiol 590: 973-989, 2012). We show that these responses exhibit continued plasticity during the 1-mo recovery period following acute spinalization. During this early chronic phase, wind-up of SA responses returned to preinjury levels in one muscle, the ST, but remained depressed in all other muscles tested. In contrast, CAP response amplitudes, which were initially potentiated following acute transection, returned to preinjury levels in all muscles except for Srt, which continued to show marked increase. These findings illustrate that spinal elements exhibit considerable plasticity during the recovery process following spinal injury and highlight the importance of considering SA and CAP responses as distinct phenomena with unique underlying neural mechanisms.NEW & NOTEWORTHY This research is the first to assess temporal summation, also called wind-up, of muscle reflexes during the 1-mo recovery period following spinal injury. Our results show that two types of muscle reflex activity are differentially modulated 1 mo after spinal cord injury (SCI) and that spinal reflexes are altered in a muscle-specific manner during this critical period. This postinjury plasticity likely plays an important role in spasticity experienced by individuals with SCI.

Motoneuron intrinsic properties, but not their receptive fields, recover in chronic spinal injury.

Proper movement execution relies on precise input processing by spinal motoneurons (MNs). Spinal MNs are activated by limb joint rotations. Typically, their movement-related receptive fields (MRRFs) are sharply focused and joint-specific. After acute spinal transection MRRFs become wide, but their manifestation is not apparent, as intrinsic excitability, primarily resulting from the loss of persistent inward currents (PICs), dramatically decreases. PICs undergo a remarkable recovery with time after injury. Here we investigate whether MRRFs undergo a recovery that parallels that of the PIC. Using the chronic spinal cat in acute terminal decerebrate preparations, we found that MRRFs remain expanded 1 month after spinal transaction, whereas PICs recovered to >80% of their preinjury amplitudes. These recovered PICs substantially amplified the expanded inputs underlying the MRRFs. As a result, we show that single joint rotations lead to the activation of muscles across the entire limb. These results provide a potential mechanism for the propagation of spasms throughout the limb.

Developmental changes in conjunctiva-associated lymphoid tissue of the rabbit.

PURPOSE: Organized conjunctiva-associated lymphoid tissue (O-CALT) is constantly exposed to environmental antigens and plays a central role in the common mucosal immune system. This study was undertaken to investigate whether O-CALT in rabbit lymphoid tissue, as in other lymphoid tissue in other mammals, changes with age. METHODS: Fluorescence stereomicroscopy was used to measure the number and size of conjunctival follicles stained with propidium iodide in rabbits ranging in age from 2 days to 57 months. To assess the function of M cells, an antigen-sampling cell type found in the follicle-associated epithelium, transcytosis of fluorescent latex beads was evaluated with confocal microscopy. RESULTS: O-CALT was not present in rabbits at birth, but appeared less than 24 hours after eyes opened at approximately day 11. The number of follicles increased with age until adolescence (2-4 months), when the number stabilized through early adulthood (17-20 months). In aged rabbits (47-57 months), there was a dramatic decline in the number of follicles. This disappearance was most pronounced in the superior conjunctiva. Average follicle diameter increased with age, except in the superior conjunctiva of aged rabbits, where the few remaining follicles were generally smaller. The uptake of latex beads showed that M-cell function was similar in all age groups. CONCLUSIONS: Age-related changes in rabbit O-CALT are similar to those that have been reported for the human conjunctiva. Preferential uptake of latex beads by follicle-associated epithelium indicates that the presence and function of M cells are not affected by aging. The lower level of O-CALT in young and elderly animals, however, would be expected to decrease their ocular mucosal immune responses.

Development of a quantitative method for evaluation of the electroencephalogram of rats by using radiotelemetry.

The objective of this study was to develop a method for recording the electroencephalogram (EEG) of rats by using radiotelemetry followed by automated analysis of the raw tracing by fast Fourier transformation (FFT). We surgically implanted four 4- to 5-month-old Wistar (Crl:(WI)BR) rats each with two epidural electrodes and an attached radiotransmitter (Physiotel Implant, model TA11CTA, Data Sciences International, Inc., St. Paul, Minn.). Raw EEG signals were routed to a digital storage oscilloscope (DataSys 7200, Gould Instrument Systems, Valley View, Ohio) with FFT and averaging capabilities. FFTs of raw signals were generated at baseline (predose) and after intraperitoneal dosing of the rats with atropine (30 min postdose; 6 mg/kg), caffeine (90 and 150 min postdose; 30 mg/kg), ketamine (15 and 30 min postdose; 50 mg/kg), and pentobarbarbital (60 and 90 min postdose; 40 mg/kg). For atropine, caffeine, and pentobarbital, the overall direction of the change in power across the spectrum (0 to 40 Hz) was the same as that reported previously. Relative peaks in power were consistent with those previous reports for atropine at < or =5 Hz and caffeine at approximately 7.5 Hz. Ketamine caused a shift in peak power from 5 to 10 Hz to < 5 Hz, as shown by other investigators. FFT analysis of EEG signals captured by radiotelemetry may provide a meaningful method for studying the CNS effect of novel compounds.