Activated caspase detection in living tissue combined with subsequent retrograde labeling, immunohistochemistry or in situ hybridization in whole-mounted lamprey brains

Abstract

In the lamprey brain, there are 18 pairs of identified spinal-projecting neurons whose regenerative abilities have been characterized. The “bad-regenerating” neurons show a very delayed form of apoptosis after axotomy (Shifman et al., 2008). Theoretically, this should provide a long window of opportunity to intervene therapeutically, so it would be helpful if we could identify the early stages of this process in vivo. Until now, there has been no method to link mRNA or protein expression directly to early-stages neuronal apoptosis in vivo. Here we describe a double-labeling protocol in whole-mounted lamprey brain for simultaneous detection of early stage apoptosis, using Fluorochrome-Labeled Inhibitors of Caspases (FLICA), and either mRNA, using in situ hybridization, or protein expression, using immunohistochemistry. To improve brain preservation, the working temperature during the FLICA stage was lowered from 37°C to 4°C (Barreiro-Iglesias and Shifman, 2012). Using this method, neurofilament protein was demonstrated by immunohistochemistry in neurons previously reacted by FLICA. The method also revealed that mRNA for the receptor protein tyrosine phosphatase PTPσ is expressed selectively in FLICA-positive neurons. In addition, our study showed that a retrograde labeling technique can be used in the context of FLICA labeling. FLICA label colocalized with TUNEL staining, confirming that FLICA labeling is a reliable marker of apoptosis in lamprey brain. Our results suggested that we can combine caspase detection with other techniques in vivo to investigate the roles and mechanisms of activated caspases and other molecules in retrograde cell deaths and regenerative abilities of neurons.

The role of RhoA in retrograde neuronal death and axon regeneration after spinal cord injury

“…Using the FLICA method adapted by us for lamprey wholemounted brains (Hu et al ., 2013), control brains showed activated caspases in very few neurons (Fig. 3A, G).…”

“…3E, G). These observations are consistent with those described in our previous reports (Shifman et al ., 2008; Hu et al ., 2013; Zhang et al ., 2014). Interestingly, the increase in FLICA-positive neurons followed a similar post-TX time course as RhoA expression changes (Fig 1A, B, E, G), suggesting that RhoA might be involved in apoptosis signaling.…”

“…Brains were dissected out and the posterior and cerebrotectal commissures of the freshly dissected brains were cut open along the dorsal midline. To evaluate caspase activity in brain neurons after spinal cord transection (Hu et al ., 2013), the brains were incubated immediately at 4 °C for 1 hour in 150 µL 1 × FLICA labeling solution, diluted with phosphate buffered saline (PBS) from 150 × FLICA labeling solution (Image-iT™ Live Red Poly Caspases Detection Kit, Cat I35101, Molecular Probe™, Thermo Fisher Scientific). Afterwards, brains were washed with 1 × washing buffer on a rotator at 4 °C, 5 min × 5.…”

“…These RS neurons have heterogeneous regenerative abilities (Jacobs et al ., 1997). The “bad-regenerating” neurons often experience very delayed retrograde apoptosis after spinal cord TX (Shifman et al ., 2008; Hu et al ., 2013; Zhang et al ., 2014). This allows us to test the role of inhibiting RhoA expression on retrograde neuronal death unambiguously.…”

Reducing synuclein accumulation improves neuronal survival after spinal cord injury

“…1C, red arrows). These neurons also exhibit other signs of post-injury degeneration, including FluoroJadeC® staining (Busch and Morgan, 2012), TUNEL staining (Shifman et al, 2008), and increased levels of activated caspases (Barreiro-Iglesias and Shifman, 2012; Hu et al, 2013), suggesting a mechanism of apoptosis. In contrast, another subset of giant RS neurons typically survives (e.g.…”