Rhesus monkeys with Lyme spirochetes in the brain

Lyme disease has been mentioned in the news a lot lately. First it was a pet chimp mauling his neighbor last month in a Connecticut town. Many news accounts of the assault reported that Travis the chimp suffered from Lyme disease and had been given tea laced with Xanax to calm his erratic behavior prior to the attack. Investigators may never figure out what provoked Travis to attack Charla Nash. Was Lyme disease a factor? Maybe (although if I had to guess, it had more to do with a wild animal being kept as a pet, living where it didn't belong).

This past weekend a human reportedly suffering from long-term Lyme disease gunned down a pastor and injured several parishioners in an Illinois church. As with any topic having to do with chronic Lyme disease, experts disagree on whether Lyme disease can trigger aggression (but I'm pretty sure what the shooter's defense attorney believes). One prominent researcher even questions whether the alleged shooter truly had Lyme disease.

A blogger on Forbes.com doubts whether chimps could be stricken with Lyme disease. In reality, seeing a chimp with Lyme disease should not surprise us at all since another nonhuman primate, the rhesus monkey, has been used for years to study neuroborreliosis, the form of Lyme disease caused by infection of the nervous system. Neuroborreliosis is observed in up to 15% of untreated Lyme disease patients. I will present two studies in which scientists examined infection of the central nervous system of rhesus monkeys by the Lyme disease spirochete, Borrelia burgdorferi.

In a 2000 study by Cadavid and colleagues, the investigators found small numbers of spirochetes in the meninges and spinal cord nerve roots of rhesus monkeys four months following laboratory infection with B. burgdorferi by needle inoculation into the skin (see figure below). In humans, the inflammatory response to the spirochetes in the meninges and nerve roots can cause meningitis (headache, stiff neck, and sensitivity to light) and radiculoneuritis (shooting pains and abnormal skin sensations), respectively.

Figure 2b from Cadavid et al., 2000. A single B. burgdorferi cell in the anterior nerve root stained by immunohistochemistry with anti-B. burgdorferi antibodies.

The investigators found no convincing evidence for the presence of B. burgdorferi in the brain parenchyma of the infected rhesus monkeys. B. burgdorferi was not found in the brain by microscopy. On the other hand, B. burgdorferi DNA was detected in brain tissue by polymerase chain reaction (PCR). This result may suggest that the spirochete is present in the brain at low levels, although the authors believed that spirochetes in the meninges surrounding the brain was the real source of the DNA. It's also possible that the N40 B. burgdorferi strain used by the authors was incapable of invading the brain of rhesus monkeys and that another strain if injected into the skin would have found its way to the brain.

Two conditions associated with neuroborreliosis disrupt brain function in humans. The first is Lyme encephalopathy, which can cause fatigue and problems with concentration and memory. Brain activity is clearly affected in patients with Lyme encephalopathy, yet the underlying pathogenic mechanism remains an enigma. Many patients with this potentially disabling condition do not exhibit the classic signs of central nervous system inflammation such as the presence of white blood cells in their cerebral spinal fluid (CSF), production of antibodies against the infective agent in the CSF, or abnormal brain MRIs. Some neuroborreliosis experts have proposed that these cases of Lyme encephalopathy are a result of "toxic-metabolic" effects of infection elsewhere in the body (such as Lyme arthritis in the joints) spilling into the brain. Nevertheless, a low-level infection of the brain by B. burgdorferi cannot be ruled out. More obvious brain involvement is observed in neuroborreliosis patients with encephalitis, in whom brain lesions are detected by MRI, accompanied by white blood cells and production of anti-B. burgdorferi antibodies in the CSF. Symptoms include slight weakness or paralysis affecting one side of the body, spastic muscles, inability to feel sensations, and bladder dysfunction. In rare cases, patients may experience strokes or seizures. Patients with encephalitis are likely to have spirochetes in their brain, usually near blood vessels.

Lyme disease may also be associated with psychiatric illness, particularly depression. Rare cases of panic attacks, bipolar disorder, mania, obsessive-compulsive disorder, dementia, and violent outbursts have also been reported.

A recent study by Mario Philipp's group in Tulane, published recently in The American Journal of Pathology, described the potential effects of B. burgdorferi on brain cell activity of rhesus monkeys. Spirochetes injected into the skin with a needle or by tick bite are unable to make it into the brain of rhesus monkeys, as I explained above. Therefore, the investigators injected live B. burgdorferi spirochetes directly into the right side of the brains of rhesus monkeys. After two weeks, they found that an average of ~10% of oligodendrocytes at the injection sites had undergone death by apoptosis, as detected by fluorescent TUNEL staining (see figure below). In contrast, fewer than 2% of oligodendrocytes underwent apoptosis when sites in the left side of the brain of the same animals were injected with saline. Oligodendrocytes produce the myelin sheaths that surround the axons of neurons and promote transmission of electric impulses; one would expect the loss of oligodendrocytes to affect neuron function.

The in vivo results corroborated ex vivo experiments in which brain slices from rhesus monkeys were incubated with B. burgdorferi for up to eight hours. In the ex vivo experiments, apoptosis of both oligodendrocytes and neurons were detected by fluorescent TUNEL staining and with antibody against activated caspase 3, a more specific marker of apoptosis. The authors speculated that neurons undergoing apoptosis in vivo were removed by phagocytes and therefore went undetected in the brain injection experiment. The other major brain cell types, microglial cells and astrocytes, were spared from apoptotic death both ex vivo and in vivo.

Figure 4A from Ramesh et al., 2008. Oligodendrocytes are stained red with antibody to S-100. Nuclei of cells undergoing DNA fragmentation, a consequence of apoptosis, are TUNEL stained green. When the images are merged, the nuclei of oligodendrocytes undergoing apoptosis become yellow. B. burgdorferi, which are stained in blue, do not appear to be in physical contact with the oligodendrocytes.

Figure 4B from Ramesh et al., 2008. Percentage of oligodendrocytes undergoing apoptosis from 3 sites of spirochete (A-C) and saline (D-F) injections (click on image for larger view).

The Tulane group also detected myriad cytokines and chemokines such as IL-6, IL-1ß, and CXCL13 being produced in the B. burgdorferi-treated brain slices by immunofluorescence antibody staining and DNA microarray analysis. The authors concluded that B. burgdorferi invasion of the brain elicits production of a brew of inflammatory mediators that promote apoptotic death of oligodendrocytes and neurons. It may be true that brain invasion by B. burgdorferi in cases of human neuroborreliosis leads to production of these cytokines and chemokines, as IL-6, IL-1ß, and CXCL13 have been detected in the CSF of neuroborreliosis patients in earlier studies. However, the apoptotic cells observed by the Tulane group may be the unnatural consequence of small areas of the brain being exposed to extremely large numbers of spirochetes. Each site was injected with 5,000 spirochetes, a number far greater than the rare spirochetes observed by microscopy in the brains of neuroborreliosis patients. For the ex vivo experiments, a 2-mm section of brain was incubated with 20 million spirochetes, again a large burden of bacteria. More convincing would be finding apoptosis of oligodendrocytes and neurons in brain tissues in patients with Lyme encephalitis. Clearly more studies are needed to understand the events that really occur when B. burgdorferi invades the brain.

References

Ramesh, G., Borda, J., Dufour, J., Kaushal, D., Ramamoorthy, R., Lackner, A., & Philipp, M. (2008). Interaction of the Lyme Disease Spirochete Borrelia burgdorferi with Brain Parenchyma Elicits Inflammatory Mediators from Glial Cells as Well as Glial and Neuronal Apoptosis American Journal Of Pathology, 173 (5), 1415-1427 DOI: 10.2353/ajpath.2008.080483

Diego Cadavid, Tim O'Neill, Henry Schaefer, and Andrew R. Pachner (2000). Localization of Borrelia burgdorferi in the Nervous System and Other Organs in a Nonhuman Primate Model of Lyme Disease Laboratory Investigation, 80 (7), 1043-1054