Search

Background and Objective Techniques that allow targeted, micrometer-scale disruption in the depths of biological tissue, without affecting overlying structures or causing significant collateral damage, could potentially lead to new surgical procedures. We describe an optical technique to make sub-surface incisions in in vivo rodent brain and characterize the relationship between the cut width and maximum depth of these optical transections as a function of laser energy. Materials and Methods To produce cuts, high intensity, femtosecond laser pulses were tightly focused into and translated within the cortex, through a craniotomy, in anesthetized rodents. Imaging of stained brain slices was used to characterize cut width and maximum cutting depth. Results Cut width decreased exponentially as a function of depth and increased as the cube root of laser energy, but showed about 50% variation at fixed depth and laser energy. For example, at a laser energy of 13 µJ, cut width decreased from 158 ± 43.1 µm (mean ± standard deviation) to 56 ± 33 µm over depths of approximately 200–800 µm, respectively. Maximal cut depth increased logarithmically with laser energy, with cut depths of up to 1 mm achieved with 13 µJ pulses. We further showcased this technique by selectively cutting sub-surface cortical dendrites in a live, anesthetized transgenic mouse. Conclusions Femtosecond laser pulses provide the novel capacity for precise, sub-surface, cellular-scale cuts for surgical applications in optically scattering tissues. Lasers Surg. Med. 43:382–391, 2011. © 2011 Wiley-Liss, Inc.

The Bombyx mori sex determination gene Bmdsx is alternatively spliced in the male and female to produce the male- and female-specific proteins. In an effort to better understand the mechanism of the alternative splicing regulation of Bmdsx, we conducted a gel-shift assay followed by LC-MS/MS analysis to identify the putative proteins bound to the cis-element CE1+6 in the exon 4 of Bmdsx. A protein named as BmHrp28 which is homologous to the Drosophila Hrp48, a member of the hnRNPA/B family, was identified and expressed in Escherichia coli for testing RNA-protein binding in vitro. All of the results showed that BmHrp28 specifically bound to the CE1+6 RNA probe. BmHrp28 has two RNA recognition motifs at the N-terminal and a glycine-rich motif at the C-terminal. It might be one of the factors involved in the male-specific splicing of Bmdsx.

Summary Purpose: Optical recording of intrinsic signals provides the highest combined spatial and temporal resolution with broad spatial sampling for measuring cerebral blood volume (CBV) and hemoglobin oxygenation in cerebral cortex. Few opportunities arise to apply this laboratory method to record spontaneous seizures in unanesthetized human brain during neurosurgery. We report such a rare opportunity in a man with recurrent focal epilepsy arising from a cavernous malformation. Methods: We recorded intrinsic optical signals (IOS) from human cortex intraoperatively during spontaneous seizures arising from brain surrounding a small cavernous malformation in an awake patient using only local anesthesia with simultaneous electrocorticography. The IOS was recorded at two wavelengths, one an isosbestic point for hemoglobin to measure CBV (570 nm) and the other at a wavelength more sensitive to deoxygenated hemoglobin (Hbr) (610 nm). A modified Beer-Lambert calculation was used on two separate but similar seizures to approximate changes in Hbr, CBV as well as oxygenated hemoglobin (HbO2). Results: Electrographically recorded seizures (n = 3) elicited a focal increase in both Hbr and CBV that lasted for the duration of the seizure, indicating that perfusion was inadequate to meet metabolic demand. Remarkably, these hemodynamic changes preceded the onset of the seizures by ∼20 s and occurred focally over the known location of the lesion and the seizure onsets. Discussion: These findings demonstrate that the hemoglobin becomes deoxygenated in spite of large increase in CBV during spontaneous human focal seizures and that optically recorded hemodynamic events can be used both to predict and localize human focal epilepsy. Such data may someday be useful to assist in the presurgical evaluation of patients considered for epilepsy surgery and to predict the timing and location of seizure onsets.

Transcriptional profiling (TP) offers a powerful approach to identify genes activated during memory formation and, by inference, the molecular pathways involved. Trace eyeblink conditioning is well suited for the study of regional gene expression because it requires the hippocampus, whereas the highly parallel task, delay conditioning, does not. First, we determined when gene expression was most regulated during trace conditioning. Rats were exposed to 200 trials per day of paired and unpaired stimuli each day for 4 days. Changes in gene expression were most apparent 24 h after exposure to 200 trials. Therefore, we profiled gene expression in the hippocampus 24 h after 200 trials of trace eyeblink conditioning, on multiple arrays using additional animals. Of 1,186 genes on the filter array, seven genes met the statistical criteria and were also validated by real-time polymerase chain reaction. These genes were growth hormone (GH), c-kit receptor tyrosine kinase (c-kit), glutamate receptor, metabotropic 5 (mGluR5), nerve growth factor-beta (NGF-beta), Jun oncogene (c-Jun), transmembrane receptor Unc5H1 (UNC5H1), and transmembrane receptor Unc5H2 (UNC5H2). All these genes, except for GH, were downregulated in response to trace conditioning. GH was upregulated; therefore, we also validated the downregulation of the GH inhibitor, somatostatin (SST), even though it just failed to meet criteria on the arrays. By during situ hybridization, GH was expressed throughout the cell layers of the hippocampus in response to trace conditioning. None of the genes regulated in trace eyeblink conditioning were similarly affected by delay conditioning, a task that does not require the hippocampus. These findings demonstrate that transcriptional profiling can exhibit a repertoire of genes sensitive to the formation of hippocampal-dependent associative memories.