Lina M.E. Pettersson

Endogenous BDNF regulates induction of intrinsic neuronal growth programs in injured sensory neurons

Identification of the molecule(s) that globally induce a robust regenerative state in sensory neurons following peripheral nerve injury remains elusive. A potential candidate is brain-derived neurotrophic factor (BDNF), the sole neurotrophin upregulated in sensory neurons after peripheral nerve injury. Here we tested the hypothesis that BDNF plays a critical role in the regenerative response of mature rat sensory neurons following peripheral nerve lesion. Neutralization of endogenous BDNF was performed by infusing BDNF antibodies intrathecally via a mini-osmotic pump for 3 days at the level of the fifth lumbar dorsal root ganglion, immediately following unilateral spinal nerve injury. This resulted in decreased expression of the injury/regeneration-associated genes growth-associated protein-43 and Talpha1 tubulin in the injured sensory neurons as compared to injury plus control IgG infused or injury alone animals. Similar results were observed following inhibition of BDNF expression by intrathecal delivery of small interfering RNAs (siRNA) targeting BDNF starting 3 days prior to injury. The reduced injury/regeneration-associated gene expression correlated with a significantly reduced intrinsic capacity of these neurons to extend neurites when assayed in vitro. In contrast, delayed infusion of BDNF antibody for 3 days beginning 1 week post-lesion had no discernible influence on the elevated expression of these regeneration-associated markers. These results support an important role for endogenous BDNF in induction of the cell body response in injured sensory neurons and their intrinsic ability to extend neurites, but BDNF does not appear to be necessary for maintaining the response once it is induced.

Markedly reduced chronic nociceptive response in mice lacking the PAC1 receptor

The neuropeptide pituitary adenylate cyclase activating polypeptide (PACAP) has been proposed to have a role in nociception. Here we have used the formalin test, thermal laser stimulation and mechanical von Frey stimulation to investigate possible alteration of PAC1−/− mice nociceptive behaviour. Our finding, that PAC1−/− mice have a substantial, 75% decrease in nociceptive response during the late phase, provides clear evidence that the specific PACAP-receptor PAC1 is involved in the mediation of nociceptive responses during chronic conditions such as inflammation. PAC1−/− mice had small or no changes in the response to mechanical and thermal laser stimulation. This suggests a limited, if any, involvement of PAC1 in nociception after short-lasting stimuli. Injury-induced changes in DRG neuropeptide expression were more pronounced in PAC1−/− mice, implying neuroregulatory functions of PAC1.

Expression of orphanin FQ/nociceptin and its receptor in rat peripheral ganglia and spinal cord.

Expression of the neuropeptide orphanin FQ/nociceptin (OFQ/N) and its receptor, the opioid receptor-like receptor (ORL1), have been found to have a wide distribution in the central nervous system, and in brain areas involved in sensory perception in particular. The effects of OFQ/N on, e.g., sensory transmission are very complex, and a modulatory effect on pain perception has been suggested. We therefore wanted to investigate the distribution of OFQ/N and ORL1 in the spinal cord and DRG, and also in SCG and some other peripheral tissues. The methods used were in situ hybridization, immunohistochemistry and ligand binding. We found that OFQ/N and ORL1 mRNA are expressed in DRG; primarily in small and large neurons, respectively. In spinal cord, mRNA for OFQ/N and ORL1 is expressed in neurons in laminae I, II and X, and in ventral horn neurons. Further, immunoreactivity for OFQ/N is observed in fibers and neurons in the superficial laminae of the dorsal horn and around the central canal, and also in neurons in the ventral horn of the spinal cord. Receptor ligand binding to the spinal cord grey matter is demonstrated, primarily concentrated to the dorsal horn and around the central canal, and also to medium and large size DRG neurons. These findings on the morphological distribution pattern of OFQ/N and ORL1 at the cellular level may support the notion that OFQ/N is involved in modulating pain transmission. Further, expression of OFQ/N and ORL1 mRNA was also found in SCG, whereas expression was undetectable in skin.

PACAP mRNA is expressed in rat spinal cord neurons

This study examines the expression of pituitary adenylate cyclase activating polypeptide (PACAP) mRNA in the rat spinal cord during normal conditions and in response to sciatic nerve transection. Previously, PACAP immunoreactivity has been found in fibers in the spinal cord dorsal horn and around the central canal and in neurons in the intermediolateral column (IML). Furthermore, in the dorsal root ganglia, PACAP immunoreactivity and PACAP mRNA expression have been observed preferentially in nerve cell bodies of smaller diameter terminating in the superficial laminae of the dorsal horn. However, neuronal expression of PACAP mRNA in adult rat spinal cord appeared limited to neurons of the IML. By using a refined in situ hybridization protocol, we now detect PACAP mRNA expression in neurons primarily in laminae I and II, but also in deeper laminae of the spinal cord dorsal horn and around the central canal. In addition, PACAP mRNA expression is observed in a few neurons in the ventral horn. PACAP expression in the ventral horn is increased in a population of large neurons, most likely motor neurons, both after distal and proximal sciatic nerve transection. The proposed role of PACAP in nociception is strengthened by our findings of PACAP mRNA‐expressing neurons in the superficial laminae of the dorsal horn. Furthermore, increased expression of PACAP in ventral horn neurons, in response to nerve transection, suggests a role for PACAP in repair/regeneration of motor neurons. J. Comp. Neurol. 471:85–96, 2004.

Nanowire Biocompatibility in the Brain - Looking for a Needle in a 3D Stack.

We investigated the brain-tissue response to nanowire implantations in the rat striatum after 1, 6, and 12 weeks using immunohistochemistry. The nanowires could be visualized in the scar by confocal microscopy (through the scattered laser light). For the nanowire-implanted animals, there is a significant astrocyte response at week 1 compared to controls. The nanowires are phagocytized by ED1 positive microglia, and some of them are degraded and/or transported away from the brain.

An array of highly flexible electrodes with a tailored configuration locked by gelatin during implantation-initial evaluation in cortex cerebri of awake rats.

Background: A major challenge in the field of neural interfaces is to overcome the problem of poor stability of neuronal recordings, which impedes long-term studies of individual neurons in the brain. Conceivably, unstable recordings reflect relative movements between electrode and tissue. To address this challenge, we have developed a new ultra-flexible electrode array and evaluated its performance in awake non-restrained animals. Methods:An array of eight separated gold leads (4 × 10 μm), individually flexible in 3D, were cut from a gold sheet using laser milling and insulated with Parylene C. To provide structural support during implantation into rat cortex, the electrode array was embedded in a hard gelatin based material, which dissolves after implantation. Recordings were made during 3 weeks. At termination, the animals were perfused with fixative and frozen to prevent dislocation of the implanted electrodes. A thick slice of brain tissue, with the electrode array still in situ, was made transparent using methyl salicylate to evaluate the conformation of the implanted electrode array. Results: Median noise levels and signal/noise remained relatively stable during the 3 week observation period; 4.3–5.9 μV and 2.8–4.2, respectively. The spike amplitudes were often quite stable within recording sessions and for 15% of recordings where single-units were identified, the highest-SNR unit had an amplitude higher than 150 μV. In addition, high correlations (>0.96) between unit waveforms recorded at different time points were obtained for 58% of the electrode sites. The structure of the electrode array was well preserved 3 weeks after implantation. Conclusions: A new implantable multichannel neural interface, comprising electrodes individually flexible in 3D that retain its architecture and functionality after implantation has been developed. Since the new neural interface design is adaptable, it offers a versatile tool to explore the function of various brain structures.

Changes in expression of PACAP in rat sensory neurons in response to sciatic nerve compression

In the present study, expression of pituitary adenylate cyclase‐activating polypeptide (PACAP) in rat dorsal root ganglion (DRG) neurons and sciatic nerve following experimental sciatic nerve compression was studied with the use of quantitative immunohistochemistry and in situ hybridization. Previously, we have investigated changes in PACAP expression after nerve transection and, here, the far more frequently encountered condition of nerve compression injury is examined. Nerve compression was performed unilaterally on the rat sciatic nerve, at mid‐thigh level, by application of a narrow silicone tube around the nerve for 3, 7, 14 or 28 days, respectively. We detect a statistically significant upregulation in the number and density of PACAP mRNA expression in both small and large DRG neurons in response to nerve compression. An increased number of PACAP‐immunoreactive neurons is also found in the ipsilateral DRG. In addition, PACAP immunoreactivity is observed in the compressed sciatic nerve segment and adjacent nerve tissue after nerve compression. The present findings can be compared with previous studies where we have shown that PACAP expression is upregulated in DRG; in response to peripheral inflammation (primarily in small–medium neurons), and after axotomy (dramatic upregulation in medium–large neurons). In view of the recent findings of an increased PACAP expression in DRG after nerve compression, as well as the previous findings of a modulation of PACAP expression in response to axotomy and inflammation, it is likely that PACAP is also involved in the modulation of the response to peripheral nerve compression.

Size-dependent long-term tissue response to biostable nanowires in the brain

Nanostructured neural interfaces, comprising nanotubes or nanowires, have the potential to overcome the present hurdles of achieving stable communication with neuronal networks for long periods of time. This would have a strong impact on brain research. However, little information is available on the brain response to implanted high-aspect-ratio nanoparticles, which share morphological similarities with asbestos fibres. Here, we investigated the glial response and neuronal loss in the rat brain after implantation of biostable and structurally controlled nanowires of different lengths for a period up to one year post-surgery. Our results show that, as for lung and abdominal tissue, the brain is subject to a sustained, local inflammation when biostable and high-aspect-ratio nanoparticles of 5 μm or longer are present in the brain tissue. In addition, a significant loss of neurons was observed adjacent to the 10 μm nanowires after one year. Notably, the inflammatory response was restricted to a narrow zone around the nanowires and did not escalate between 12 weeks and one year. Furthermore, 2 μm nanowires did not cause significant inflammatory response nor significant loss of neurons nearby. The present results provide key information for the design of future neural implants based on nanomaterials.

Multiple implants do not aggravate the tissue reaction in rat brain.

Chronically implanted microelectrodes are an invaluable tool for neuroscientific research, allowing long term recordings in awake and behaving animals. It is known that all such electrodes will evoke a tissue reaction affected by its’ size, shape, surface structure, fixation mode and implantation method. However, the possible correlation between tissue reactions and the number of implanted electrodes is not clear. We implanted multiple wire bundles into the brain of rats and studied the correlation between the astrocytic and microglial reaction and the positioning of the electrode in relation to surrounding electrodes. We found that an electrode implanted in the middle of a row of implants is surrounded by a significantly smaller astrocytic scar than single ones. This possible interaction was only seen between implants within the same hemisphere, no interaction with the contralateral hemisphere was found. More importantly, we found no aggravation of tissue reactions as a result of a larger number of implants. These results highlight the possibility of implanting multiple electrodes without aggravating the glial scar surrounding each implant.

Injury-Associated PACAP Expression in Rat Sensory and Motor Neurons Is Induced by Endogenous BDNF

Peripheral nerve injury results in dramatic upregulation in pituitary adenylate cyclase activating polypeptide (PACAP) expression in adult rat dorsal root ganglia and spinal motor neurons mirroring that described for the neurotrophin brain derived neurotrophic factor (BDNF). Thus, we posited that injury-associated alterations in BDNF expression regulate the changes in PACAP expression observed in the injured neurons. The role of endogenous BDNF in induction and/or maintenance of PACAP mRNA expression in injured adult rat motor and sensory neurons was examined by intrathecally infusing or intraperitoneally injecting BDNF-specific antibodies or control IgGs immediately at the time of L4-L6 spinal nerve injury, or in a delayed fashion one week later for 3 days followed by analysis of impact on PACAP expression. PACAP mRNA in injured lumbar sensory and motor neurons was detected using in situ hybridization, allowing quantification of relative changes between experimental groups, with ATF-3 immunofluorescence serving to identify the injured subpopulation of motor neurons. Both the incidence and level of PACAP mRNA expression were dramatically reduced in injured sensory and motor neurons in response to immediate intrathecal anti-BDNF treatment. In contrast, neither intraperitoneal injections nor delayed intrathecal infusions of anti-BDNF had any discernible impact on PACAP expression. This impact on PACAP expression in response to BDNF immunoneutralization in DRG was confirmed using qRT-PCR or by using BDNF selective siRNAs to reduce neuronal BDNF expression. Collectively, our findings support that endogenous injury-associated BDNF expression is critically involved in induction, but not maintenance, of injury-associated PACAP expression in sensory and motor neurons.